Jessie, alias Musashi, serves as a core Team Rocket operative, consistently partnered with James and Meowth. Her primary mission focuses on capturing powerful or rare Pokémon, especially Ash Ketchum's Pikachu, though her schemes routinely fail. She channels a highly self-centered and dramatic personality, frequently prioritizing her own whims and desire for the spotlight above Team Rocket's wider objectives. This manifests as arrogance, underestimating opponents, and a short temper prone to furious outbursts triggered by plan failures or criticism of her appearance—particularly insults about her age or damage to her long magenta/fuchsia pink hair and beauty. Despite this volatility, she possesses a strong sense of pride and a fierce rivalry, notably with fellow agent Cassidy. Her signature attire is a customized Team Rocket uniform: a short white-sleeved shirt bearing a large red "R" on the chest with high-neck collars, layered over a black short-sleeved shirt revealing her midriff, paired with a white miniskirt, black thigh-high boots, green pearl-like earrings, and long black arm-sleeves. Jessie excels at disguise, adopting varied outfits for infiltration, including formal wear with glasses and a bun, athletic wear, or student disguises featuring elements like purple tunics, striped leggings, and dyed hair.

Her relationship with her Pokémon is complex and occasionally contradictory. She demonstrates clear preferences for specific types, particularly snake-like Pokémon like Ekans/Arbok and Seviper, along with Poison-types. While she can be harsh, shouting at or physically striking her Pokémon in frustration after defeats, she also exhibits genuine care and sacrifice. Key instances include tearfully releasing her Dustox so it could pursue romance, helping her Arbok and James's Weezing escape by shielding a group of Ekans and Koffing from an antagonist's Pokémon attacks, diligently repairing her Mimikyu's torn disguise using her sewing skills despite its initial disobedience, and cherishing her Gourgeist—fondly reminiscing about its care as a Pumpkaboo and defending it post-incident. Her Pokémon roster across regions included Shellder (briefly), Lickitung (traded for Wobbuffet), Woobat, Yanmega (replacing Dustox), Frillish (noted for strong moves but underused), Seviper (a long-term Hoenn/Sinnoh companion), and Mimikyu (her sole Alolan capture, initially disobedient but eventually bonding). Battle proficiency varied; Pokémon like Yanmega possessed strong move sets and speed, while others like Lickitung or Gourgeist showed limited effectiveness.

During the Unova arc (Black & White series), Jessie underwent a shift upon promotion to Advanced Agent, adopting a more serious, calm, and collected "true villain" persona while retaining her underlying mean streak. This professional demeanor subsided after the thwarting of "Operation Tempest," leading to a reversion to her classic temperamental self. Later, in the XY series, she developed romantic feelings for Dr. White after he saved her and Wobbuffet, working as his assistant under the alias "Jessilia" until discovering his affections lay elsewhere. She also pursued fame, aspiring to become the Kalos Queen and achieving temporary recognition before losing to Serena. Additional traits include being a shopaholic, capable of spending Team Rocket's funds excessively when encountering shopping malls.