BackgroundNumerous studies have been carried out to investigate the factors that influence the human immunodeficiency virus (HIV) knowledge of college students. However, the impact of cultural attitudes towards sexuality, particularly those related to masturbation, on the HIV knowledge within this population has not been comprehensively explored. The present study aimed to examine the factors influencing the HIV knowledge of Chinese college students, with a particular focus on its association with the cultural attitudes regarding masturbation. MethodsA cross-sectional study was conducted via the widely used Wen-Juan Xing questionnaire platform. A total of 6350 students participated anonymously, among which 5945 with complete responses formed the final sample. This sample consisted of 47.5% males and 52.5% females, with an average age of 19.35 +/- 1.28 years. Independent sample t-tests, chi-square tests, and multiple linear regression analyses were utilized for data analysis. ResultsStudents with lower masturbation beliefs scale scores (LMBF), signifying less conservative beliefs and greater acceptance of masturbation, demonstrated better knowledge about HIV (t = 16.980, p = 0.000) compared to those with higher scores. Moreover, the LMBF group exhibited higher accuracy on the brief HIV knowledge questionnaire (HIV-KQ-18) questionnaire (p < 0.05). Multivariate linear analysis indicated that age and the masturbation belief scores were negatively correlated with the HIV-KQ-18 scores. Students in higher grades surpassed freshmen in terms of their performance on the HIV-KQ-18, and those from wealthy families scored higher. Respondents who were female, came from villages, belonged to multi-child families, had a sexual history, were heterosexual, and had no history of sexually transmitted infections (STIs) had significantly lower scores in HIV knowledge (p < 0.05). The overall correction rate of the HIV-KQ-18 was 62.0% among college students. ConclusionsCollege students had a rather limited understanding of HIV, which is influenced by multiple factors such as age, sex, the place of residence during childhood, year of study, family income, sexual history, sexual orientation, and the history of STIs. At the same time, cultural attitudes towards masturbation also had a particular influence on college students' knowledge of HIV. Consequently, it is essential to introduce customized HIV education programs to improve college students' comprehension and rectify cultural misconceptions regarding sexuality.