To assess trends in HIV-1 incidence and risk factors for seroconversion among men who have sex with men (MSM) resident in Rome, Italy, a retrospective longitudinal cohort study was conducted over 25 years. Incidence rates and trends were modelled using Poisson regression and risk factors were assessed by multivariate Cox models. Of 1,862 HIV-1-negative individuals, 347 seroconverted during follow-up. HIV-1 incidence rates increased from 5.2/100 persons/year (p/y) in 1986 (95% confidence interval (CI): 2.3–11.5) to 9.2/00 p/y in 1992 (95% CI: 6.4–13.0), decreased to 1.3/100 p/y in 2001 and increased until 2009 (11.7/100 p/y; 95% CI: 7.4–18.6). The risk of HIV-1 seroconversion increased during the study period in younger MSM (incidence rate ratio (IRR) = 17.18; 95% CI: 9.74–30.32 in 16–32 year-olds and IRR = 5.09; 95% CI: 2.92–8.87 in 33–41 year-olds) and in those who acquired syphilis (IRR = 7.71; 95% CI: 5.00–11.88). In contrast, the risk of seroconversion decreased among highly educated MSM (IRR = 0.54; 95% CI: 0.35–0.82) and those without Italian citizenship (IRR = 0.45; 95% CI: 0.28–0.71). The HIV epidemic in MSM living in Rome continues to expand. Targeted prevention programmes against sexually transmitted infections to enhance knowledge transfer and behavioural skills are urgently required.