A striking feature of insulin expression is its almost complete restriction to beta-cells of the pancreatic islet in normal mammals. Here we show that insulin is expressed in and secreted from human ejaculated spermatozoa. Both insulin transcript and protein were detected. In addition, the large differences in insulin secretion, assessed by RIA, between noncapacitated and capacitated sperm suggest a role for insulin in capacitation. Insulin had an oscillatory secretory pattern involving glucose dose-dependent increases and significant decreases during the blockage of an insulin autocrine effect. It appears that the effect of glucose on the fertilizing ability of sperm is mediated by glucose metabolism through the pentose phosphate pathway. Then we evaluated the autocrine effect of sperm insulin on glucose metabolism by studying the activity of glucose-6-phosphate dehydrogenase, the key rate-limiting enzyme in the pentose phosphate pathway. The simultaneous decrease in both insulin release and glucose-6-phosphate dehydrogenase activity induced by blocking the autocrine insulin effect with three different procedures (blockage of insulin release by nifedipine, immune neutralization of the released insulin by antiinsulin serum, and blockage of an insulin intracellular effector such as phosphotidylinositol 3-kinase by wortmannin) strongly suggests a physiological role of sperm insulin on these two events. Insulin secretion by spermatozoa may provide an autocrine regulation of glucose metabolism based on their energetic needs independent of systemic insulin. In conclusion, these data open a new area of study in male reproduction.