Winnie-the-Pooh: Rabbit and Piglet (both male) are often mistaken to be female by those not familiar with the franchise. Even those who are big fans often think that way despite knowing better.
Rabbit's voice sounds like it could be a high-pitched man or a low-pitched woman (all of his voice actors have been male), he's Ambiguously Gay (wears a pink bathrobe, curlers, frilly aprons and can ballet dance) and is Super OCD.
Piglet also has a high-pitched voice wears a pink swimsuit-like outfit and has a shy and submissive personality. His American voice actors have all been male, but some of his foreign ones were female.
The original voice actor for Piglet was John Fiedler (possibly best known as Mr. Peterson, the older, balding, patient on The Bob Newhart Show), who did have a rather breathy and high-pitched voice. Subsequent American voice actors have mostly tried to imitate Fiedler's voice (Fiedler himself died in 2005).
One of the strangest examples is the title character. In the book, not only is the toaster explicitly without gender, but this is something of a plot point when the group encounters a mated pair of squirrels that cannot conceive of a genderless being (the toaster ultimately convinces both of them separately that it is the same gender as they —male for the male and female for the female —but really it's just to spare their feelings). For the film, however, fans are pretty evenly split between "the Toaster is male" and "the Toaster is female". A few lines of dialogue suggest explicitly that Toaster is male: firstly, Lampy says that "He sank" in the waterfall scene; secondly, Radio refers to Toaster and Blanky as "you boys" at the beginning of the journey; and thirdly, some of the song lyrics refer to the five characters as "the new boys in town." In addition, promotion material and trailers (such as this one) refer to the Toaster as male. Nevertheless, Toaster's personality is fairly gender-neutral, and thanks to Cross-Dressing Voices, the fact that Toaster's voiced by a woman is no help. And we're still talking about a machine. Ultimately, the toaster can be viewed as a gender-neutral character by those who want to. The voice acting, actions, attitudes, fears, thoughts, and character development do not suggest any particular gender, but don't suggest It's Pat-style androgyny or a robot either. The Slippy Toad demeanor and voiceover begs the question, but the only reason you'd consider the toaster's gender is if you think about it years after watching, or if someone else brings up the question.
Who really can be mistaken for female is Flower, the skunk. When he is a child, his voice and speech patterns sound nothing like a male, he acts shy and bashful, is very fluffy, and spends much of his time smelling flowers. A couple years after his introduction, when Bambi meets him again, his voice has deepened and he gets an only slightly more effeminate girlfriend. There is the picture book of the Disney movie that actually called Flower a female, and made "her" amother!
Bambi himself is also a subject to this when he's a child. As an adult he's quite obviously male, though. Bambi II did hang a lampshade on this when a young Ronno called Bambi "Princess" as an insult.
The makers of Monsters VS Aliens themselves seem to be confused over the gender of Insectosaurus. A behind-the-scenes book labels the creature as a "she" while, within the canon of the actual film, the monster is a "he". However, given that he/she is the only monster that can't speak, the latter could be a case of Your Tomcat Is Pregnant, as the Tertiary Sexual Characteristics that cause the confusion are only revealed when he/she metamorphs into a butterfly at the end of the film.
The twin dolls 3 and 4 in 9 look completely identical (save for their stamped-on numbers), act alike, and never speak, making it completely ambiguous as to what gender either of them is. This is compounded by the fact that, as living ragdolls, they don't have any external characteristics indicative of sex. The creator has urged fans to come up with their own theories regarding the two.
The starfish, Peach, in Finding Nemo. Its voice actor is a woman, but her voice is just masculine enough to make it sound like a guy. Conceivably intentional, as some varieties of starfish are hermaphroditic.
Terk (voiced by Rosie O'Donnell) is frequently mistaken for male despite her mom stating otherwise in the first minutes of the film. This has been lampshaded by Tantor in The Legend of Tarzan. Adding to the confusion, the stage musical adaptation made Terk a male.
A large number of viewers of trailers for Kung Fu Panda, who were unaware of just whom Angelina Jolie had been cast as, were in for a shock when they finally saw the film and Master Tigress spoke for the first time. In everyone's defense, not only did the lack of Tertiary Sexual Characteristics make it very hard to tell her gender, even her voice actress originally assumed, when the casting call was made public, that Jackie Chan would be playing the tiger. This confusion only added to the Stupid Sexy Flanders associated with the character (although for some viewers it didn't lessen the appeal).
Not many people would have thought Kazuma from Summer Wars was a guy, given both his hair-style, Cross-Dressing Voice and Vague Age, which all fits somewhat perfectly for a Gamer Chick stereotype. His online avatar is even a rabbit (however, this rabbit is a badass).
Two of the background race cars from Cars: According to their toy bios, the pink race car sponsoring Tank Coat is actually male, and the blue race car sponsoring RevNGo is actually female.
Remember the alligator from All Dogs Go to Heaven? You always thought it was a female, right? WRONG. He's male. It doesn't help that he's never mentioned by name in the film (only in tie-in materials). Otherwise, the fact that his name is King Gator would be a huge hint to his gender. His singing voice is also very masculine, but could be heard as a deep female voice too.
Rolly from 101 Dalmatians. In the animated series and his cameo in House of Mouse, he's been given a blue collar. Only female puppies wear blue, the boys all wear red. Oddly he's the only male to be given a blue collar and he's still clearly referred to as male.
Doris from the Shrek is actually female. For those of you who did already know that, it is pretty easy to mistake her for being male, and manyoldarticles refer to her as 'he'.
The Incredibles has Edna Mode, or "E", who first-time viewers sometimes mistake for a guy. This can mostly be attributed to her not having the traditional Tertiary Sexual Characteristics, or a noticeable figure; not to mention being voiced by the film's director, Brad Bird, for extra androgynous points.
Cupcake from Rise of the Guardians. She wears pink clothes and a skirt, and is obsessed with horses and unicorns, but looks more like a boy than a girl, and can easily be mistaken for one.
Rancis Fluggerbutter. He's one of the few boys among the Sugar Rush racers, his hair is long enough to be mistaken for a girl's pixie cut, and his name is based off of the name Francis, which is a boy's name, but which sounds identical to the name Frances, which is a girl's name.
Swizzle Malarkey is also, surprisingly, a guy. He has long hair and there are more female Sugar Rush racers than there are male, so some fans have gotten confused.
How to Train Your Dragon accidentally gave male Toothless the dragon some feminine symbolism. He regurgitates some food for the main character Hiccup like a mother bird. In a scene where he looks at a bird's nest, the intention was to show how Toothless wanted to fly, but the camera lingered on the eggs for a bit too long, so it seemed like Toothless wanted to get back to his own eggs rather than wanting to fly. The way he holds onto Hiccup at the climax of the film resembles a mother and her newborn child. To be fair, in many bird species, males participate in taking care of the young just as much, if not more, than females (and in some species, "sex role reversal" appears and males take over parental care entirely from females). This could as well be the case for Night Fury dragons.
The baby Lady's owners have in Lady and the Tramp always wears pink and has a very pink room with bows on their crib. He is however a boy and it wasn't uncommon in The Gay '90s for boys to wear pink. The sequel film, which was created in the 2000s, altered his design so that he wears blue instead of pink.
Mei from Arashi no Yoru ni is an exceedingly adorable little goat who sometimes wears a pink handkerchier. He is paired with the much larger Gabu, who is a scruffy looking wolf. The entire film has them running away together to escape their clan's judgement on their friendship. It's not uncommon for people to mistake him for a girl, especially since it's common to see the film as a romance, though Mei has an unmistakably adult male voice. Mei was in fact gender neutral in the original books, which caused a lot of fans to assume he was a girl until the movie adaptation. In contrast the All-CGI Cartoon has him as a female goat.
The protagonist Tykvenok of the 1984 Soyuzmultfilm short cartoon And what can you do? (а что ты умеешь?) is a pumpkin with visible eyelashes, a flower on his head ( Later lost it after he grew up to save his friends, but was given more flowers on his head at the end), and was voiced by veteran voice actress Clara Rumyanova (she provides any number of other cases throughout her career), yet he is always referred to as male. Actually, the Russian word for "pumpkin" is female, but creating female forms for the young is a bit problematic.
Littlefoot from The Land Before Time could easily be mistaken for a female due his somewhat feminine appearance and voice, soft spoken and polite personality, and eyelashes even though he's usually voiced by a boy. Ostinably made a bit better with the introduction of Ali in the fourth film, who was explicitly colored pink to basically say "I'm female".
In the 1972 animated adaptation of Pinocchio, Un Burattino Di Nome Pinocchio one could easily mistake the titular character as a puppet for a girl, due to having a female voice actor in both the original Italian and English dubs, having a somewhat feminine looking face, and wearing a pink bonnet and a coat that looks like a dress. However he looks much more masculine when he becomes a real boy.
To especially young children who overlook her womanly figure and "body language," the gender of Ursula in The Little Mermaid is sometimes a point of confusion, due to her large size, shortish hair and deep alto voice. Of course she was modeled after the drag queen Divine. In the live-action theatrical version Ursula is nearly always played by a male actor.
Batbayar from The Cave of the Yellow Dog movie has odd-looking pigtails probably make him seem more like a little girl to Most Western audiences.
Destoroyah from the Godzilla franchise was never officially given a gender, but is considered male by most fans. However, that hasn't stopped other fans from thinking he's a female because some aspects of his body structure are similar to that of female crabs.
And, then there's also Mothra Leo from the Mothra Film Trilogy. For the record, Leo is a boy. Yes, you heard me. A Male Mothra (Mothrus?) despite every single incarnation previous and since being female.
Even the dub of the films had trouble with this and constantly referred to Leo as both a "he" and a "she" and even an "it".
Mothra herself has been mistakenly referred to as a male in some works, although generally not those officially licensed by Toho and more often in works referencing her.
An interesting variation of this occurs with the monster Rodan. In the original 1956 film Rodan, both a male and a female Rodan are featured. Since then, fans have argued whether or not the Rodans(?) featured in later films are male or female.
Some people have mistaken Gigan, a male cyborg kaiju, for a female due to his high-pitched roar.
Battra, Mothra's Evil Twin, has also been mistaken for a female; even in officially licensed works.
And it doesn't end there, with Kroiga from Latitude Zero. With the brain of a human female, but the body of a male lion.
Kumonga has been mistaken to be a female by the fandom. This is due to the dub of Son Of Godzilla not using any gender-specific pronouns regarding the giant spider. The original Japanese dub, on the other hand, refers to the monster as a male.
Kamacuras is also officially a male, but fans often mistake him for a female. However, since there's more than one Kamacuras, it could be possible at least one or more of them is a girl.
Some fans have mistaken Manda as a female kaiju due to some works branding him as such.
Godzilla himself, sort of. See, suit actor Haruo Nakajima believes that the Showa Era (1955-1975) Godzilla is a female while the original 1954 monster was a male. However, Toho Studios has stated that all the incarnations of Godzilla that we've seen are male. Considering that the films establish that there is more than one Godzilla, and that infants like Minilla and Junior show that there's a breeding population, the idea of a female Godzilla isn't that far fetched. It's just that we haven't officially seen a lady Godzilla yet.
There are people out there who think the Newborn from Alien: Resurrection is a female. For the record, it's a hermaphrodite. The actual prop used for the film had both male and female genitalia. This actually applies to all members of the Xenomorph species, even the Queen. H.R. Giger intended the Aliens to be neither male nor female, but an unsettling blur of both sexes.
Some viewers of The Year of Living Dangerously were surprised to learn that Billy Kwan was played by a woman, Linda Hunt. She's the only actor to receive an Academy Award for playing a character of a different sex.
Jodie Foster's daughter in Panic Room (played by the then 11-year old Kristen Stewart) has a fairly gender neutral haircut, somewhat androgynous looks and wears uni-sex pyjamas and can easily be mistaken for a boy on first viewing.
A lot of viewers of Fatal Attraction think that Ellen Gallagher (played by child actress Ellen Hamilton Latzen) is a boy because of her short hair and androgynous face. There are frequent heated debates on The Internet Movie Database about whether they "should have cast a girl that looked like a girl". It doesn't help that she is seen rehearsing for a school play and playing a male role. Also, some of the adult characters tend to pronounce her name as if they're saying "Alan" instead of "Ellen".
Sam the German shepherd isn't revealed to be female until she gets hurt, at which point Will Smith calls her by her full name of Samantha.
The daughter Marley, played by Willow Smith, is this for some viewers. Whenever we see her she has a fairly short hairstyle, an androgynous name and gender neutral clothes. It isn't actually said whether or not she's a girl until about an hour into the film. Many people also assume that she's a male as a deliberate reflection of the survivors Anna and her young companion Ethan, reinforced when Neville momentarily hallucinates them as Zoe and Marley.
In Heroic Trio, the Big Bad looks like an effeminate male but has a female voice. This confusion might only be the Western audiences who might not be as familiar with Chinese legends concerning eunuchs making for extremely powerful soldiers while simultaneously making them evil and manipulative.
Many fans thought Buckwheat from the Our Gang/Little Rascals shorts was a girl. This is because initially the character was a girl, played by Matthew (Stymie) Beard's real-life sister Carlena. Even after male actor Billie Thomas inherited the role, the character continued to be portrayed as a girl for several shorts.
Alexa in 50 First Dates, which is intentional on the part of the movie.
Robin Williams' daughter in What Dreams May Come is very androgynous, and it's perfectly possible to go through most of the movie thinking she's a boy.
Dennis, the kid who wanted pancakes, from Cabin Fever. He has very long hair, a somewhat feminine face, has a gender neutral voice, and is prepubescent from the looks of it.
Quite a few people who hadn't read the source material of The Never Ending Story mistook the warrior Atreyu to be a girl. When he was first introduced, Bastian, who was expecting an adult warrior, commented in surprise, "A little boy." Which people might have missed if they came into the movie right in the middle.
Intentionally done in The Dark Knight Rises; the audience is led to believe that the one prisoner who escaped the prison was Bane, when actually it was Talia Al Guhl. The reveal is shown when after escaping, the child wraps her head and shoulders in a burkha.
During early promotions for The Force Awakens, many fans mistakenly believed that Dark Action Girl Captain Phasma was a man. The problem being that in the promotional materials initially given, she didn't speak and wasn't seen without her armor on.
The cobra in Jungle Book has a feminine voice but is referred to as male. It can be disassociating, especially since Kaa has a deep voice in comparison.
Leslie from the made-for-TV film of Bridge to Terabithia. She has an androgynous look and a Gender-Blender Name. This is intentional and even Jess in the books mistakes her for a boy, but this isn't noted in the film so many viewers think she's a boy. Fans of the 2007 film (where Leslie was given a huge Girliness Upgrade) often ask why Leslie is a boy in the '80s film.
Oscar from Icebox is a preteen boy with shaggy, medium-length hair. This makes him androgynous looking to some viewers.
Jack from the film adaption of Room due to having long hair. He's actually mistaken for a girl when he escapes to the real world for the first time.