Even better, beating Mega Satan with this power up. Enjoy killing Mega Satan by peeing onto his face! Doubles as a Crowning Moment of Awesome, obviously.
Isaac can wear his mother's brassiere or her menstrual pad as items. Doing so causes the enemies to freeze or back away, respectively. Presumably, they're Squicked out by the sight of it!
Segmented enemies like Larry Jr. will even fall apart when they try to flee. They are so disgusted that it breaks the laws of physics.
When you meet Greed in a hidden room, you can immediately leave through the hole you made without having to defeat him. Hi, Greed. Bye, Greed. If you have X-ray glasses, you can even use his room as a shortcut, running past him without ever engaging him.
Sadly no longer works in Rebirth, since the entrances to the rooms around the hidden room get blocked until you defeat him.
The beggar that runs the shell game in the arcade is a jerk. Giving you a giant grin as he takes all your money without giving you the items you want? Seeing the slot machine throw a troll bomb at him is just too hilarious a karma to be true.
The glitches in this game can sometimes cause hilarious reactions from players. Especially since, given the tone of the game and previous games by Edmund McMillen, it's difficult to tell if some of them are actually glitches.
One of the random effects of pills is "I Found Pills" with the description "...and ate them." This pill also causes Isaac to get a silly, derped look on his face and make a faint "duuur" noise when you take it.
Another effect of the pills is the "Puberty" pill, which causes Isaac to grow a few scraggly hairs on the top of his head. Then you realize, those are pubes growing on his head...
Even more amusing pills in the remake. A new pill effect is "R U A Wizard?" This causes Isaac to get a derpy, wall eyed look that forces him to shoot his tears diagonally for a short while.
The fact that the game treats this as a positive pill effect makes it even funnier.
Any of the secret messages from the Fortune Telling machine. Some of these include cryptic nonsense like "LOOK TO LA LUNA" or "DON'T LEAVE THE HOUSE TODAY". Then there are some that seem to be talking to the player, such as "GO OUTSIDE" or "YOU ARE THROWING YOUR LIFE AWAY". The way the game is just totally fucking with you in this moment is priceless. Oh, the best of all these? YOU ARE WORSHIPPING A SUN GOD.
Some of the boss enemies make some pretty funny expressions, such as Chub's poker face during her intro, War's Oh, Crap! face when he runs out of stamina, Pin's stereotypical frown (it looks exactly like ":c"), or even Loki's expressions of pure glee while he fires his 4-way shots.
The fact that of all the Seven Deadly Sins, the only one without a unique sprite is Sloth. Or, in more modern terms, laziness.
One of the between-level dreams features Isaac using the toilet... and discovering there's no toilet paper left. His reaction is the typical "No!" reaction used in other dreams, but it seems more like he's going "Curses!" this time around. Don't worry, Isaac, other people can relate.
In Afterbirth, one of the new dream transitions is Isaac and his penis falling off. His reaction is what any man's reaction would be if his dick fell off.
Fighting Wrath, a mini-boss who uses bombs... while you have the Remote Detonator on hand. Even better if you have Bobby-Bomb on hand, which pretty much means you can sit back and laugh while Wrath gets chased by his own bombs.
In Rebirth, picking up the Rosary item allows you to potentially sell your soul for the Bible.
Rebirth adds an item called "How to Jump", its description being "it's time you learned." The animation for jumping is just Isaac's head slapped onto the hopper sprite. It's as ridiculous as it sounds; it must be seen for oneself.
One secret seed you can input, BASE MENT, gives you a seemingly normal run. Basement I and Basement II are normal, but instead of moving on to Caves I, you go down to Basement III?, which is exactly like Basement I. And then Basement IV?! and Basement V...And then Basement VI and Basement VII. If you have time, you can play the seed for 4 hours!
If you make it all the way to the thousanth floor, you'll get a special message:
The red poop dropped by the Carrion Queen will damage any other enemy that touches it. Because of this, during the Boss Rush, it's possible for some bosses to die as soon as they enter the room.
Cancer, in real life, is a horrible, life-threatening disease. Cancer in The Binding of Isaac is probably one of the best trinkets in the game, since it doubles your tear-firing rate, even if you've already hit the cap. The tagline description for it in Rebirth is even "Yay, cancer!"
Made even better by the fact that you can get rid of it at any time!
One ending (ending 11 in Rebirth) has Isaac trying to open up the chest, only for It Lives! to appear, try to growl intimidatingly, and give an incredibly cheeky grin.
The very next ending (actually the previous ending in Rebirth) has ??? give Issac a cheeky grin as well. Made even funnier in the original game/WotL, where the Dramatic Chipmunk Sting plays during this scene.
The Greed Mode ending continues this gag. We find out Greed is yet another posthumous Isaac as he gives us this big dumb grin.
Afterbirth+ has the Greedier Mode ending following the above with his head falling off and a literal geyser of spiders erupting from the severed neck. Because why not.
He's the number one. God's favorite son. His flowing robes reach down from heaven. His hands will touch you! Build a world around you! The light within his heart is enough to heal us all! He will fill your soul when life takes its toll. Your only goal is to find his kingdom. His love is real! The golden seal! Trust in the book and the bread you took!
And under the main post, there's a bunch of strikethrough text that details the many other changes that will be made.
Note: other minor changes made to the 3ds and wiiU release are as follows: isaacs blood has been replaced with sweat. all instances of pills have been replaced with lollipops. all demon forms are now purple instead of black. you can now choose the gender identity of eden when you choose to play as them. maggy is now Hispanic. the womb has been replaced with a meat boy themed world. all instances of suicide now have text popups of local suicide hotlines. the letters R P A and E have been removed from the games code. isaacs mother is no longer overweight unless you choose the fat acceptance mode from the options menu, you can also replace mom with isaacs dad or isaacs dog from this menu. all references to isaacs sexuality being anything other than straight have been removed and he now carries a football. isaac is no longer shamed for dressing slutty. mulligans now wear police hats. any instance of abortion has been replaced with a funny cat video. the devil is no longer black. Isaac no longer eats dog foot and spoiled milk, but healthy snacks of apples. the A button has been replaced with the “Trigger Button” a simple tap when triggered will close the game and removed it from your system. Isaac is now 18. racism no longer exists and finally all instances of my name have been removed from the game as well as any association with my blog, past games and this post.
The Adversary's design. For the record, The Adversary is the post-mortem version of the Dark One, and all of the other undead bosses have suffered some form of Body Horror (Monstro II is missing most of his skin, the Carrion Queen is almost completely skeletal, etc). The Adversary? Covered in bandages and missing a horn. It almost looks comical.
When the achievements for Afterbirth were posted on Steam slightly before release, those with Real Platinum God saw that they no longer had 100% of achievements. Instead, they had 69%. Whether or not this is intentional is unknown, but likely.
Two of the pills from Afterbirth, named "One Makes You Larger" and "One Makes You Small", make you bigger and smaller, respectively. They also stack, so you can either end up with an Isaac who can barely even be seen he's so small, or an Isaac who takes up half of the screen with his size. It needs to be seen to be believed.
Ultra Greed's rotating sprites include several of his butt. This has reached the point of Memetic Mutation and causes things like this to pop up.
The Chaos item from Afterbirth. After picking this item up, all further collectible items in the run will be drawn from random item pools, which can have... interesting effects. Such as an Angel showing up and offering you Butter Bean or a Devil Room offering the Halo. And then there's the expression◊ Isaac gets after picking up the item...
Upon finding Bum Friend, Dark Bum, and Key Bum in one run, a cutscene will trigger, showing the three merging together to become Super Bum.
The Epilogue that plays after beating Mom for the first time starts off with her about to kill Isaac...then a Bible hits her in the head.
Rita: That wasn't a fight... *beat* That was a mess.
The Aprils Fool challenge in Afterbirth +. Nothing is what it looks like, every boss is the Bloat unless you randomly use Book of Revelations (every active item is Dead Sea Scrolls on steroids, so have fun if you get an infinite use item like Guppy's Paw), and the reward is Maggy starting with a pill.
To make this even funnier, a Reddit user once suggested a challenge where every boss is The Bloat. When Afterbirth+ came out (and with it the Aprils Fool challenge), they were banned from /r/FUCKBLOAT (the official anti-Bloat subreddit) and forced to use a unique "I Love The Bloat" flair on the main Isaac subreddit.
The Binning of Isek: Afterberts. To say any more would spoil it. It's a graphical overhaul mod that turns every graphic into a poorly-drawn MS Paint picture, turns all the music into awful midis, and all the text is misspelled. And yet, it's 100% playable. It's every bit as incredible as it sounds.
The icon for the 1000000% achievement? A glittering stop sign with a gold background. The message? "Just Stop!"
Delirium can shapeshift into other bosses, and if it rolls a boss with multiple health-based forms, its form will adapt to its current health. This means that if Delirium transforms into Brownie at low health, it kills itself as the aforementioned boss splits into Dank Squirts when its health gets low, resulting in a hilarious anti-climax.