Josephine "Jo" March, second eldest daughter of the March family, lives near Gettysburg during the American Civil War. Her father's military absence strains the household finances, leading Jo to seek work. She first assists her stern great-aunt, Marta March, who disapproves of Jo's unconventional ways. Later, Jo secures a position at a Newcord newspaper, aspiring to publish her fiction despite facing criticism from editor Anthony.

Defying societal expectations of femininity, Jo exhibits a tomboyish and outspoken demeanor marked by a fierce temper. This often sparks conflict with her youngest sister, Amy, culminating in Amy burning Jo's manuscripts out of spite—a rift temporarily severing their bond. Reconciliation comes when Jo rescues Amy after she falls through thin ice while following Jo and Laurie. Jo's protective nature extends fiercely to her frail sister Beth; she nurses Beth through a near-fatal scarlet fever contracted while aiding an impoverished family. Though Beth recovers, she is left permanently weakened, an outcome that deeply affects Jo.

Financially pragmatic, Jo sells her long hair—her "one beauty"—to fund her mother's urgent trip to Washington when Mr. March falls seriously ill, rejecting offers of aid from Aunt March and Mr. Laurence. Her family loyalty intertwines with literary ambition; she primarily writes stories to entertain Beth, who serves as her muse. Following Beth's decline and death, Jo experiences a creative block, burning old manuscripts before channeling her grief into writing a serious novel dedicated to Beth.

Jo forms a close friendship with Theodore "Laurie" Laurence, their neighbor's grandson, sharing adventures and secrets. Laurie develops romantic feelings, but Jo rejects his marriage proposal, viewing him solely as a brother. He subsequently departs for Europe with his grandfather. While working as a governess in New York, Jo meets Professor Friedrich Bhaer; he critiques her sensationalist writing, and though mutual respect develops, no romantic conclusion is depicted in the anime.

Her career evolves from pulp fiction to completing a novel inspired by her family. A publisher insists the heroine marry for commercial success, but the anime emphasizes Jo's focus on literary achievement. In the final episodes, she relocates to New York with Anthony to pursue journalism, leaving romantic resolutions ambiguous. The narrative reveals Laurie eventually marries Amy, becoming Jo's brother-in-law.

Physically, Jo is tall and thin with gray eyes, a "funny-looking" nose, and initially unmanageable long brown hair, later cut short. Her movements are often awkward, resembling a "colt." As she matures, her appearance softens; she gains curves and carries herself with increased grace.