Josephine Mailstar appears only in the OVA episode "Jeffrey's Knighthood." She requests sorceresses Lina Inverse and Naga the Serpent to transform her son Jeffrey into a knight worthy of the Royal Guard. A misunderstanding arises when her phrasing suggests she wants them to engage sexually with Jeffrey, which she angrily corrects; her actual intent is for Jeffrey to perform heroic deeds and build his reputation.

As Jeffrey's overprotective mother and a member of the noble Mailstar family, Josephine secretly follows her son on his adventures, concealing herself with a bag over her head. Her defining trait is extreme protectiveness, manifested by physically striking anyone who criticizes Jeffrey or points out his shortcomings, typically using a large mallet. She attacks Naga multiple times for such remarks. Despite these overt interventions, Jeffrey remains completely unaware of her presence, mistakenly crediting himself for successes.

Josephine orchestrates Jeffrey's training by hiring actors to pose as easily defeated bandits. When real bandits capture these actors, Lina and Naga must intervene. Later, Josephine hires the entire "Flaming Knights" mercenary group to support Jeffrey against a warlord's beast-man army. Her interventions go beyond planning; she bursts unexpectedly into combat, like emerging from the ground, to defeat enemies insulting her son. The climax reveals her confronting her husband, Baron Goldias, as the mastermind behind the conflict opposing Jeffrey. She attacks Goldias with her mallet, resolving the situation. Josephine exhibits no significant character development beyond her combative and manipulative maternal role. Her background, motivations, and relationships receive no deeper exploration. She appears in no other Slayers media beyond this single OVA episode.