The purpose of this study was to examine the impact of prolactin (PRL) on human sperm function, in light of a recent proteomic analysis indicating that these cells express the PRL receptor (PRLR). Immunocytochemical analyses confirmed the presence of PRLR in human spermatozoa and localized this receptor to the postacrosomal region of the sperm head as well as the neck, midpiece, and principal piece of the sperm tail. Nested PCR analysis indicated that these cells possess four splice variants of the PRLR: the long form and three short isoforms, one of which is reported for the first time. A combination of Western blot analyses and immunocytochemistry demonstrated that PRL inhibited sperm capacitation in a dose-dependent manner, suppressing SRC kinase activation and phosphotyrosine expression, two hallmarks of this process. The suppression of sperm capacitation was accompanied by a powerful prosurvival effect, supporting the prolonged motility of these cells and preventing the formation of spontaneous DNA strand breaks via mechanisms that involved the concomitant suppression of caspase activation. Western blot analyses indicated that the prosurvival effect of PRL on human spermatozoa involved the stimulation of Akt phosphorylation, whereas inhibitors of phosphatidylinositol-3-OH kinase and Akt negated this effect, as did the direct induction of sperm capacitation with cAMP analogues. We conclude that PRL is a prosurvival factor for human spermatozoa that prevents these cells from defaulting to an intrinsic apoptotic pathway associated with cell senescence. These findings have implications for preservation of sperm integrity in vivo and in vitro.