Before becoming a ROOT ninja, Sai was bald and had a British accent.
Kuki is voiced by Lauren Tom, who plays Amy and her mom, Inez, on Futurama, Tasumi on The Replacements, Connie (Kahn, Jr.) and her mom, Minh, on King of the Hill. And in continuing with being vocally typecast as an East Asiannote Chinese, Japanese, Korean, etc girl and her mother in cartoons, Tom plays both Kuki Sanban and her mother, Genki in this series.
Numbuh 3 is also Jinx. Mushi gets to play her in Jinx's last episode. In fact, one of the only young, popular characters she voices that doesn't fit into the Asian Airhead trope in some manner is Kahn "Connie" Souphanousinphone, Jr. from King of the Hill.
Numbuh Four is voiced by Dee Bradley Baker in one of the rare times that Baker isn't voicing an animal (Klaus the goldfish from American Dad), doing announcer work on a game show (like Shop Til You Drop and Legends of the Hidden Temple), or doing animal vocal effects. He's also voiced the Gravemind.
Numbuh Three was originally only going to speak Japanese with an occasional English subtitle.
Numbuh Five originally was supposed to be The Voiceless (this was only fulfilled in "Operation: T.H.E.-F.L.Y.).
"Operation: S.P.A.N.K.E.N.S.T.E.I.N." was originally a thirty-minute Thanksgiving special.
Also, Lizzie's original character design was changed before her debut episode was made. Apparently her old appearance was too ugly for the execs.
One episode, "Operation: T.R.E.E.S.", an episode-long Shout Out to Dr. Seuss and specifically to The Lorax, was axed early in its production, due to it running right on the heels of the movie of Horton Hears a Who!.
The Toiletnator was originally going to be a one-time character in Operation N.O.-P.O.W.U.H. only.
Grandfather was originally going to have a wife shown!