This study was designed to evaluate potential preventive effects of a cultural adaption of the Focus on Kids (FOK) program among Chinese adolescents through a quasi-experimental intervention trial in Nanjing, China. High school students were assigned to either experimental groups (n = 140) or control groups (n = 164) by schools (with three schools in each condition). The participants completed a confidential questionnaire at baseline and 6-month post-intervention with a follow-up rate of 94.4% (287 of 304). The outcome measures included HIV knowledge, HIV-related perceptions based on the protection motivation theory, stigmatizing attitude toward people living with HIV/AIDS (PLWHA), intentions of health-related risk behaviors and sexual intercourse in the previous 6 months. Results showed a significant intervention effect at 6-month post-intervention in increasing HIV knowledge, decreasing perceptions of response cost associated with abstinence and reducing stigmatizing attitudes toward PLWHA, after controlling for key demographic characteristics and relevant baseline measures. Further mediation analysis suggested that HIV knowledge mediated the effect of intervention on stigma reduction. Findings from this study support the feasibility and initial efficacy of the cultural adaptation of FOK HIV prevention program among high school students in China.