The series centers around the Bone Family, white, bald cartoon caricatures with big noses. In the opening pages the three Bone cousins—avaricious Phoncible P. "Phoney" Bone, goofy cigar-smoking Smiley Bone, and everyman character Fone Bone—are run out of their hometown of Boneville after Phoney decides to run for mayor and built a balloon [thought to be an evil god later in the story] on top the head of a statue of Boneville's founder. The balloon broke the head off of the statue and all the towns people ran Phoncible, Smiley, and Fone out of town. After crossing a desert and ending up in the mysterious Valley, the cousins are separated by a sea of locusts, and must individually make their way across the fantasy landscape pursued by rat creatures. They joyously reunite at a local village called Barrelhaven, where they are taken in by a mysterious girl named Thorn and her even more enigmatic grandmother. Fone Bone instantly develops a crush on Thorn when he meets her, and repeatedly attempts to prove his love through poetry. As they stay longer in the valley, they encounter humans and other creatures who are threatened by a decrepit dark lord, the Lord of the Locusts. The Bones are quickly drawn into the events around them, compelling them on a hero's journey to help save the world.

Although Boneville is never actually shown in the story, it is implied as technologically contemporary: Fone refers to its extensive downtown, Phoney carries dollar bills, and Smiley refers to the presence of nuclear reactors and a CornDogHut. In contrast, the Valley is depicted as somewhat medieval, judging by its lifestyle, use of a bartering system, weapons and modes of transportation, and Phoney persistently refers to the valley people as " yokels."

Although essentially a high fantasy, Bone also displays slapstick humor, particularly in The Great Cow Race (issue #10) and Phoney Bone's ongoing efforts to become rich off the credulous valley residents. As the series progresses to graver issues and a more serious level, its characteristic use of broad humor lessens but continues to recur.