Two enzymes are involved in the polyol pathway: an aldose reductase that reduces glucose in sorbitol followed by its oxidation in fructose by sorbitol dehydrogenase. It has been previously shown that both enzymes are presented in the bovine epididymis, where they are associated with membranous vesicles called epididymosomes. Based on the distribution of these enzymes, it has been hypothesized that the polyol pathway can modulate sperm motility during the epididymal transit. In the present study, polyol pathway was investigated in semen and along the epididymis in humans in order to determine if sperm maturation can be associated with this sugar pathway. Western blot analysis shows that both aldose reductase and sorbitol dehydrogenase are associated with ejaculated spermatozoa and prostasomes in humans. These enzymes are also associated with epididymosomes collected during surgical vasectomy reversal. Western blot, Northern blot, and reverse transcription-polymerase chain reaction analysis show that aldose reductase and sorbitol dehydrogenase are expressed at the transcriptional and translational levels along the human epididymis. Unlike what occurs in the bovine model, distribution of these enzymes is rather uniform along the human excurrent duct. Immunohistological studies together with Western blot analysis performed on epididymosomes preparations indicate that the polyol pathway enzymes are secreted by the epididymal epithelium. These results indicate that the polyol pathway plays a role in human sperm physiology.