The number of new syphilis cases in Brazil has risen alarmingly in recent years. However, there is limited data regarding syphilis prevalence in the Brazilian prison population. To facilitate the development of effective interventions, a cross-sectional study was undertaken to determine the prevalence of Treponema pallidum infection, active syphilis, and associated risk factors among Brazilian prisoners. We administered a questionnaire to a population-based sample of prisoners from 12 prisons in Central-West Brazil and collected sera for syphilis testing, from January to December 2013. Univariable and multivariable regression analyses were performed to assess associations with active syphilis. We recruited 3,363 prisoners (men: 84.6%; women: 15.4%). The overall lifetime and active syphilis prevalences were 10.5% (9.4% among men; 17% among women, P < 0.001) and 3.8% (2% among men; 9% among women, P < 0.001), respectively. The variables associated with active syphilis in men prisoners were homosexual preference, history of sexually transmitted infections, and human immunodeficiency virus status. Among women, the factors were sex with intravenous drug users, genital ulcer disease, and previous incarceration. Despite the high prevalence of active syphilis, 88.5% reported unawareness of their serological status and 67% reported unprotected sexual practices. Women had the highest rates of infection, including them in a high-risk group for the development of syphilis during pregnancy. Thus, implementing screening programs to enable continuous measures of control and prevention of T. pallidum infection in the prison environment, mainly in women institutions, is important to prevent severe forms of this disease and congenital infections.