We have previously demonstrated that the amount of HE1/NPC2 mRNA and protein expressed in the human epididymis is decreased under vasectomy. In this study, western blot analyses showed that many vasovasostomized men are characterized by high HE1/NPC2 levels in spermatozoa when compared with fertile donors. HE1/NPC2 association with sperm from vasovasostomized men was not related to low motility per se as spermatozoa from asthenospermic men have HE1/NPC2 levels similar to those in normal fertile semen samples. Spermatozoa from vasovasostomized men with high amount of HE1/NPC2 are characterized by higher concentration of cholesterol and more lipid raft domains. HE1/NPC2 is secreted in different glycoforms by different tissues of human male reproductive tract. These forms are due to variation in N-glycosylation, and only the deglycosylated form is associated with spermatozoa from some vasovasostomized men. Compared with normal men, seminal plasma of vasectomized men is characterized by a major decrease in immunodetectable HE1/NPC2 without change in the glycosylation pattern. Following surgical vasectomy reversal, seminal plasma HE1/NPC2 was found in similar amounts to the ones characterizing normal men. Considering the potential role of HE1/NPC2 in cholesterol transport during sperm maturation, unusual high levels of this protein associated with spermatozoa of vasovasostomized men may reflect epididymal sequelae occurring when the vas deferens is obstructed.