This study aims to explore the psychological mechanisms underlying high school students’ attitudes toward homosexuality, with a focus on the influence of gender. A structural equation model was constructed based on the “Gender–Cognition–Affect–Behavior” framework. Previous studies have mostly focused on the relationships between background variables and attitudes, while paying relatively little attention to the internal psychological processes involved. As discrimination against homosexuality still exists in school settings, understanding the formation and expression of such attitudes is particularly important. Based on the CAB model, this study adopted a mixed-methods design that combined quantitative and qualitative approaches, integrating structural equation modeling (SEM) with interview analysis. A total of 163 valid samples were collected from first-year high school students. The results indicated that cognition did not have a significant direct effect on behavior, whereas affect played a mediating role between cognition and behavior. In addition, gender had a significant effect on affect, with male students exhibiting stronger negative emotions toward homosexuality. Overall, affect plays a key role in the process through which cognition is transformed into behavioral intention, and gender is deeply involved in this process. The findings of this study have important theoretical and practical implications for gender education and anti-discrimination interventions.