Background: The consequences of homophobia can affect the integrity, mental and physical health of homosexual individuals in society. There are few studies in Peru that have evaluated homophobia in the medical student population. Objective: To establish the social, educational and cultural factors associated with homophobia among Peruvian medical students. Methods: A cross-sectional analytical study was conducted in 12 medicine schools in Peru. Homophobia was defined according to a validated test, which was associated with other variables. Statistical associations were identified. Results: The lowest percentages of homophobic students (15–20%) were found in the four universities in Lima, while universities in the interior of the country had the highest percentages (22–62%). Performing a multivariate analysis, we found that the frequency of homophobia was lower for the following variables: the female gender (PRa=0.74; 95% CI, 0.61–0.92; p=0.005), studying at a university in Lima (PRa=0.57; 95% CI, 0.43–0.75; p<0.001), professing the Catholic religion (PRa=0.53; 95% CI, 0.37–0.76; p<0.001), knowing a homosexual (PRa=0.73; 95% CI, 0.60–0.90; p=0.003) and having treated a homosexual patient (PRa=0.76; 95% CI, 0.59–0.98; p=0.036). In contrast, the frequency of homophobia increased in male chauvinists (PRa=1.37; 95% CI, 1.09–1.72; p=0.007), adjusted by four variables. Conclusions: Homophobia was less common in women, in those who study in the capital, those who profess Catholicism and those who know/have treated a homosexual. In contrast, male chauvinists were more homophobic.