Jessie, identified as Musashi in Japanese-language media, serves as a core member of the Team Rocket trio alongside James and Meowth. Her early life involved significant hardship; raised by her mother, a Team Rocket agent who vanished on a mission to find Mew, Jessie subsequently entered foster care. There, she endured extreme poverty, with meals sometimes reduced to snow flavored with soy sauce. A childhood incident further marked her disillusionment: an attempt to capture Santa Claus resulted in her favorite doll being taken by a Jynx, causing her to stop believing in Santa. Academically, she struggled, lacking friends and failing to qualify for Pokémon nurse school. She later attended a Chansey school but could not graduate due to an inability to learn the move "Sing." A brief enrollment at Pokémon Tech saw her meet James, where both achieved the lowest recorded scores. Before joining Team Rocket, she belonged to a biker gang, earning the nicknames "Big Jess" and "Chainer Jess," and held various jobs while navigating unsuccessful relationships.

Her personality blends vanity, a volatile temper, and pride. She reacts with intense fury to insults about her age, damage to her face, or harm to her long magenta hair – incidents include a Scyther shortening her hair to shoulder-length and a Seviper biting it. She frequently asserts ownership with declarations like "Your things are mine, my things are still mine," and underestimates opponents, contributing to her failures. Despite this abrasive demeanor and her role stealing Pokémon for Team Rocket promotion, she demonstrates genuine care for her own Pokémon. She exhibits a distinct preference for Poison-type and snake-like Pokémon, such as Ekans (which evolved into Arbok), Seviper, and her released Dustox, alongside favorites like Dratini and Smoochum.

Jessie maintains a fierce rivalry with fellow Team Rocket agent Cassidy, fueled by Cassidy's perceived superiority and Jessie's own history of failures and low rank. Despite frequent disapproval from Giovanni regarding the trio's inefficiency, Jessie remains loyal to Team Rocket's objectives, undertaking missions for promotion. Her ambitions extend beyond villainy; she actively participates in Pokémon Contests and Showcases under aliases like Jessilina, Jessilie, or Jessilia, employing disguises and costumes to compete for ribbons or Princess Keys, showcasing versatility outside her criminal activities.

In *Pokémon the Movie: Volcanion and the Mechanical Marvel*, Jessie, James, and Meowth are hired by Councillor Alva of the Azoth Kingdom to capture the artificial Pokémon Magearna. They pursue Ash, Volcanion (magically chained to Ash), and Magearna during the group's journey to the Nebel Plateau. Their intervention triggers a confrontation where they attack the protagonists but are driven off with assistance from Princess Kimia and her Shiny Gardevoir. After the climax, where Volcanion seemingly sacrifices itself to destroy Alva's fortress before being revealed alive, the Team Rocket trio observes the resolution from a distance, expressing satisfaction at the outcome.

Her development includes a period where her core traits shifted; upon promotion to Advanced Agent in the Unova region, she adopted a more calm, collected, and traditionally villainous demeanor, though she reverted to her characteristic temperament after the failure of Operation Tempest. Her foundational relationship with James and Meowth is marked by mutual reliance and shared history, enabling them to pursue side jobs and hobbies alongside their Rocket duties. Meowth translates Pokémon speech for the trio, and James occasionally conceals his true feelings about her viewpoints to maintain harmony. This dynamic endures across regions as they relentlessly pursue Ash's Pikachu.