Many studies have reported the effects of cytokines on human sperm function, even though their role and the mechanisms involved remain unclear. The effects of increasing concentrations of the cytokines TNF-α and IL-6 on human sperm motility and viability were assessed, and the possible mechanisms involved were investigated. TNF-α and IL-6 significantly reduced progressive motility at higher concentrations in a dose- and time-dependent manner. No differences were observed in cell viability. Both cytokines increased nitric oxide production in a dose-dependent manner. TNF-α and IL-6 did not statistically differ in their detrimental effects on human spermatozoa. These results indicate that TNF-α and IL-6 have an effect on sperm function. This effect is possibly mediated via an increase in nitric oxide production.