Yotsuba&! is centered on the antics of Yotsuba Koiwai, a five-year-old adopted girl who is energetic, cheerful, curious, strange, and quirky—so strange and quirky, in fact, that even her own father calls her a weirdo. She is also initially ignorant about many things a child her age would be expected to know, among them doorbells, escalators, air conditioners, and even playground swings. This ignorance results in humorous stories where she learns about, and frequently misunderstands, everyday things.
At the start of the series, Yotsuba and her adoptive father, Koiwai, relocate to a new city with the help of Koiwai's best friend, an impressively tall man nicknamed Jumbo. Yotsuba makes a strong impression on the three daughters of the neighboring Ayase family, Asagi, Fuka, and Ena, and many of her misadventures come from interactions with them.
The series has no continuing plot—the focus is Yotsuba's daily voyage of discovery. Significantly, many chapters can be identified as taking place on successive days, which reinforces the sense of that we are following the characters' daily life. The tone can be summarized by the motto, used on chapter title pages and advertising, "Today is always the most enjoyable day" ("Itsu de mo kyō wa, ichiban tanoshii hi").