Background: Acquired syphilis continues to affect millions of people around the world. It is crucial to study it in the context of HIV Pre-Exposure Prophylaxis (PrEP) to achieve the goals set out in the 2030 Agenda since the literature suggests increased risk behaviors for sexually transmitted infections. This study aimed to investigate the incidence and factors associated with acquired syphilis among PrEP users. Materials and methods: This retrospective cohort included data on PrEP users from all over Brazil from 2018 to 2020, obtained from the national antiretroviral logistics system. We calculated the proportion of syphilis before PrEP, the incidence during the user’s follow-up, reinfections, and their possible associated factors. We conducted descriptive, bivariate, and multivariate analysis, estimating the crude Relative Risk, adjusted Odds Ratio (aOR), and their respective confidence intervals (95%CI). Results: Most of the 34,000 individuals who started PrEP were male (89.0%), white (53.7%), self-identified as male (85.2%), homosexual, gay, or lesbian (72.2%), and had 12 schooling years or more (67.8%). Of these, 8.3% had syphilis in the six months before starting PrEP, and 4% had it in the first 30 days of using the prophylaxis. In the 19,820 individuals effectively monitored, the incidence of acquired syphilis was 19.1 cases per 100 person-years, and 1.9% of users had reinfection. The multivariate analysis identified female gender (aOR 0.3; 95%CI 0.2-0.5), being white or Black (aOR 0.9; 95%CI 0.7-0.9 and aOR 0.7; 95%CI 0.7-0.99, respectively) as protective factors for syphilis. Being homosexual, gay, lesbian (aOR 2.7; 95%CI 2.0-3.7) or having a history of syphilis in the six months before PrEP (aOR 2.2; 95%CI 1.9-2.5) were risk factors for syphilis during PrEP use. Behaviors related to the risk of syphilis included accepting something in exchange for sex (aOR 1.6; 95%CI 1.3-1.9), irregular condom use (use in less than half of sexual intercourse sessions; aOR 1.7; 95%CI 1.53-2.1) and recreational drug use (poppers; aOR 1.5; 95%CI 1.53-2.1). Conclusion: Syphilis in the context of PrEP has high rates and is associated with sociodemographic and behavioral factors. We recommend additional studies targeting prevention in this population to curb these figures.