This study assessed the main factors associated with HIV infection among men having sex with men (MSM), in order to provide evidence for future intervention. A case-control study was conducted among HIV cases identified at the MSM survey in Nanjing, Suzhou and Yangzhou cities from May to July in 2008. Each HIV case was matched by 4 HIV negative MSM who participated consecutively right after the HIV case was identified at the same survey, same time and in the same city. A national core MSM survey questionnaire was used with addition of variables of local interest. Fitting conditional logistic regression model was applied for univariate and multivariate analysis. During the survey, 62 HIV positive cases were identified and 248 controls were matched. Data from univariate analysis showed that, inconsistent condom use, syphilis infection, self reported sexually transmitted infection (STI) symptoms and/or signs, unprotected anal sex (UAI) were risk factors, with OR values of 5.56 (95% CI: 1.82-16.96), 2.19 (95% CI: 1.18-4.05), 2.50 (95% CI: 1.13-5.50) and 3.13 (95% CI: 1.64-5.98) respectively. Online cruising and receptive anal sex were protective factors with OR values as 0.47(95% CI: 0.22-0.998) and 0.67 (95% CI: 0.23-1.97). Through multivariate analysis, it was found that syphilis, age of having experienced first insertive sexual intercourse and UAI were risk factors to HIV transmission, with OR values of 2.28 (95% CI: 1.05-4.94), 4.16 (95% CI: 1.63-10.66) and 1.11 (95% CI: 1.04-1.18) respectively. Compared with non-anal sex, bisexual (OR = 0.41, 95% CI: 0.18-0.97) behavior seemed a protected factor. Syphilis infection, UAI and self reported STI symptoms and/or signs were risk factors related to HIV transmission while bisexual seemed a protective factor.