Objective: To explore the impact of online sexuality education courses on the sexual knowledge, attitudes and related behaviors of rural girls, so as to provide the practical guidance for promoting sexual health and development. Methods: From February to June 2023, by posting information online and through commonweal organization websites, rural primary schools in 12 provinces were recruited for a semester of online sexuality education courses from October to November 2023. A self compiled sexuality education questionnaire was used to survey the knowledge, attitudes, and behaviors of rural girls before and after the course intervention, with pre tests in September 2023 and post tests in December 2023. The eligible samples were 3 058 and 2 602 , respectively. Independent sample t tests and text frequency and sentiment analysis were used to process the data Results: In terms of sexual knowledge, the scores of rural girls before and after the test were (8.49±3.29) and (9.40±3.35), respectively, with the post test score being higher than the pre test ( t =-10.20, P <0.01). In terms of attitudes, the scores of rural girls before and after the test were (11.50±4.62) and (10.82±4.80), respectively, with a decrease in stigmatization towards physiological development in the post test ( t =5.40, P <0.01). Regarding sexual related behaviors, the frequency of sexualbased bullying among rural girls was (5.12±2.13) before and (4.89±2.18) after, with a statistically significant difference ( t =3.99, P <0.01). The frequency and willingness of rural girls to discuss sexual topics with significant others both increased on post test (pre test:8.45±2.62; post test: 8.73± 2.62) and (pre test:8.90±2.46, post test:9.16±2.46), with a statistically significant difference ( t =-3.91, -4.03, P < 0.01 ). Text analysis revealed that "boys" "girls" and "menstruation" were the most concerned topics among the participants, and compared to before receiving sex education (69.91%), the proportion of negative emotions among rural girls decreased ( 18.59 %). Conclusion Online sexuality education courses can improve the sexual knowledge of rural girls, reduce stigma and negative emotions towards sex, decrease the incidence of sexual based bullying and increase the frequency and willingness to discuss sexual topics with parents, teachers, and peers.