Semen HIV-1 level is an important determinant of the risk of HIV-1 sexual transmission. In this prospective observational study, we investigated potential associations between seminal cytokine levels and HIV-1 concentration in the seminal plasma of HIV-infected men, using paired blood and semen samples from 18 HIV-1 chronically-infected men off antiretroviral therapy. HIV-1 RNA levels and cytokine levels in seminal plasma and blood were measured and analyzed using simple linear regressions to screen for an effect of cytokines on seminal plasma HIV-1. Forwards stepwise regression was performed to construct the final multivariate model. The median HIV-1 concentration was 4.42 log10 RNA copies/ml (IQR 2.98, 4.70) and 2.96 log10 RNA copies/ml (IQR 2, 4.18) in blood and semen plasma, respectively. In stepwise multivariate linear regression analysis, blood HIV-1 level (p<0.00005), seminal plasma IFN-gamma level (p=0.03), and seminal plasma IL-17 level (p=0.03) were positively associated with seminal plasma HIV-1 level; seminal plasma IL-5 was negatively associated with seminal HIV-1 level (p=0.0007). These data indicate that, in addition to plasma levels of HIV-1, cytokine profiles in the male genital tract are an important determinant of HIV-1 levels in semen. Th1 and Th17 cell profiles are associated with increased levels of virus while the Th2 cellular profile is associated with decreased levels of virus. These results support the importance of genital tract immunomodulation in HIV-1 transmission.