Holly is a substitute teacher who makes her first appearance on Glee in the second season's seventh episode, "The Substitute". She is filling in at McKinley High School for the ailing Spanish teacher Will (Matthew Morrison), who is also director of the glee club, New Directions. Club member Kurt (Chris Colfer), who had seen her perform "Conjunction Junction" when she subbed for his English class, asks her to also take over Will's glee club rehearsals. Instead of assigning songs, Holly asks the club members what kind of music they would like to perform, and when Puck (Mark Salling) suggests "Forget You", she sings the song and they all join in, except for Rachel (Lea Michele). Holly later wins her over by asking Rachel what she would like to sing that she hasn't been able to, and they perform a number from Chicago together. Holly bonds with Sue (Jane Lynch), who is the acting principal with Principal Figgins (Iqbal Theba) also out sick, and Sue fires the still-ailing Will, making Holly the full-time director of the glee club. When he recovers, Will confronts Holly at school, but she is unwilling to give up her new position. She later discovers she is in over her head when Mercedes (Amber Riley) gets in trouble, and Holly turns to Will for help. She reveals that she was once a more serious teacher like Will until a student punched her in the face, at which point she became far more free spirited. Holly ultimately returns to substitute teaching and Will is reinstated. He assigns the glee club to perform "Singin' in the Rain" on his return, but faced with their dismay at being given another old song, he asks for Holly's help to modernize it, and they all perform a mash-up of it with Rihanna's "Umbrella".
Holly later returns to McKinley High as a substitute sex education teacher in the season's fifteenth episode, "Sexy". She informs Will that the members of New Directions are woefully ignorant about sex. He invites her to teach the glee club about safe sex, and she performs a rendition of Joan Jett's "Do You Wanna Touch Me (Oh Yeah)", much to the chagrin of the new head of the celibacy club, guidance counselor Emma (Jayma Mays). Holly advises Puck and Lauren (Ashley Fink) that the sex tape they plan to make is illegal, as both are minors, and helps Brittany (Heather Morris) and Santana (Naya Rivera) start to come to terms with their true feelings for each other, later performing "Landslide" with them in front of the club. When Emma's choice of "Afternoon Delight" as an abstinence song reveals her sexual naivete, her husband Carl (John Stamos) asks Holly for an appointment for him and Emma to work out issues in their sex life. During the counseling session, he reveals that Emma is still a virgin, and in answer to a question from Holly, Emma admits that she may still have feelings for Will. At the end of the episode, Holly begins a relationship with Will.