We aimed to assess the risk of infections caused by sexual practices and determine the factors affecting the prevalence of human immunodeficiency virus (HIV) and syphilis among men who have sex with men (MSM) in Lanzhou, China. A survey including 600 MSM was conducted from March 2008 to December 2009. Demographic and sexual behavior data were collected using the snowball sampling technology, and blood samples were obtained. Multivariate logistic regression models were used to determine the predictors of HIV and syphilis infection. The seroprevalence of HIV, syphilis, HCV, and co-infection of HIV with syphilis was 6.0%, 18.0%, 1.3%, and 2.5%, respectively. Use of condoms was more inconsistent in vaginal sex than that in anal sex, buying sex or selling sex. Multivariate analysis showed that MSM with syphilis infection, MSM from other provinces, and MSM of Han ethnicity were more likely to have HIV infection. Further, MSM with HIV infection, inconsistent condom use, and less knowledge about AIDS prevention were more likely to have syphilis infection. Their social background, habit of condom use, and infection status may affect the prevalence of HIV or syphilis. These findings may have important implications for further behavioral interventions among this population.