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Homophobic and transphobic beliefs that lead to bias-based harassment remain a critical concern for young people in the USA. The aim of the present study was to examine the impact of an inclusive comprehensive sex education program (High School FLASH) on homophobic and transphobic beliefs. Data from this study come from a randomized controlled trial that evaluated the impact of High School FLASH on students' sexual behaviors and related outcomes with 20 schools in two U.S. regions (Midwest and South). Following the baseline survey, the 20 schools were randomly assigned to receive FLASH or a comparison curriculum. Ninth and 10th grade students completed follow-up surveys 3 and 12 months after the instructional period. We examined changes in homophobic beliefs using multilevel linear regression models in the full sample and two sub-groups: straight cisgender young people versus those who identified as not straight or cisgender. Mean scores on the homophobic and transphobic beliefs scale were statistically significantly lower among young people receiving FLASH relative to the comparison at both the 3- and 12-month timepoints (p-values for adjusted mean differences were < 0.01, n = 1357 and 1275, respectively). Specifically, FLASH's positive impact on reducing homophobic and transphobic beliefs was statistically significant for straight and cisgender youth at both survey follow-ups (p < 0.01, n = 1144 and p = 0.05, n = 1078, respectively); the effects for the LGBTQ sub-group reached statistical significance at only the final follow-up (p = 0.01, n = 197). Our results show that carefully designed, inclusive comprehensive sexual health education programs like High School FLASH can play a role in promoting better school climates for all youth by reducing beliefs that may lead to bullying, violence, and victimization. [ABSTRACT FROM AUTHOR]