To date, there is a lack of a structured national plan of sexual education in Italy, with large interregional differences and poor uniformity in the promotion and learning of sexual health. The aim of this study was to evaluate the level of knowledge on sexuality and reproductive function among adolescents before and after a sexual education program, considering gender differences in the results. A longitudinal and interventional study was conducted on high school students. Six sessions for each class were carried out by a multidisciplinary team. Anonymous questionnaires were administered to investigate basic knowledge of human sexuality and reproduction before and after intervention. Participants were high school students who completed the questionnaires. Outcomes included pre and posttest answers on the knowledge questionnaire in study population. We obtained data from 842 high school students (mean ± SD age, 16.11 ± 1.74years). The main informative sources on sexuality were social media in 37.9% of participants; family, school, and scientific material were the main source in 15.1%, 5.2%, and 4.4%, respectively. A total score of 12.6 ± 3.4 (range, 1-20) was obtained on the knowledge questionnaire; specifically, the score was higher in females than males (12.8 ± 3.4 vs 11.9 ± 3.4, P = .0001). In males and females, a statistically significant increase in level of knowledge was found after the multidisciplinary intervention (14.5 ± 3.6, P = .0001). The lack of sexual education programs in schools in Italy compels adolescents to refer to frequently unreliable sources, such as social media and peers, contributing to misinformation and adoption of risky sexual behaviors. This study is based on a large sample size and highlights the importance of sexual education programs. The main limitations are a greater representation of the female population and the inclusive nature of the school (ie, an artistic school), attracting students who identify with sexual minorities. Adolescents have limited knowledge about sexuality and reproduction, with a notable gender disparity; sexual education programs in schools, providing information from sexuality experts, led to a substantial increase in knowledge and a reduction in the gender gap.