"Genshiken: Second Generation" serves as a sequel to the original "Genshiken" series, continuing the story of the college club Genshiken, which is dedicated to otaku culture, including anime, manga, and gaming. The narrative shifts focus to a new generation of club members while maintaining ties to the original cast. The story begins with Madarame, a former member and now a salaryman, reconnecting with the club. The new members include Hato, a cross-dressing male artist with a passion for BL (boys' love) manga; Ogiue, now the club president and a published manga artist; and Sue, an eccentric American exchange student with a deep interest in Japanese otaku culture.

The series explores the dynamics between the old and new members as they navigate their shared interests and personal struggles. Hato's cross-dressing becomes a central point of tension and curiosity, particularly for Madarame, who struggles to understand Hato's identity and motivations. Ogiue, now more confident in her role as a leader, balances her responsibilities as president with her creative ambitions. Sue's quirky personality and cultural misunderstandings add comedic elements while highlighting the global appeal of otaku culture.

As the club engages in activities such as attending conventions, creating doujinshi (self-published works), and discussing their favorite media, the narrative delves into themes of identity, acceptance, and the evolving nature of fandom. The interactions between the characters reveal their growth and challenges, with Madarame often serving as a bridge between the old and new generations. The series maintains a balance between humor and introspection, offering a nuanced portrayal of otaku life and relationships. Through its focus on both personal and collective experiences, "Genshiken: Second Generation" provides a continuation of the original series while exploring new perspectives within the otaku community.