--- - |2 The mechanisms by which varicocele affects fertility remain undetermined. Vitamin A (all‐trans retinoic acid [ATRA]) is required for fertility and normal spermatogenesis; however, the mechanisms driving its action are not defined yet. Previously, we demonstrated in varicocele sperm a reduced RARα expression and that ATRA influence sperm performance. To further define vitamin A significance in male gamete and in the physiopathology of varicocele, we tested for the first time ATRA action on human sperm metabolism and antioxidant defense systems. Evaluating triglycerides content and lipase activity, in normal sperm ATRA had a lipid lowering effect, which was not observed in varicocele sperm. The modulation of the glucose‐6‐phosphate dehydrogenase activity, concomitantly with a reduction of the glucose content, highlight an ATRA role on glucose metabolism. ATRA induced the superoxide dismutase (SOD) and glutathione transferase activities, while it reduced the malondialdehyde and reactive oxygen species (ROS) production both in healthy and varicocele sperm. Interestingly, SOD1 and SOD2 have been localized in the acrosome and midpiece, glutathione‐ S‐transferase omega 2 (GSTO2) in the acrosome, equatorial, and subacrosomial regions. SOD1, SOD2, and GSTO2 levels were significantly lower in varicocele with respect to healthy sperm. Herein, we discovered that ATRA treatment was able to reprogram sperm metabolism toward that of the capacitation status. The retinol protected human sperm from ROS damage enhancing the antioxidant enzymes activity, providing evidence toward the efficacy of vitamin A as therapeutic tool in improving sperm quality. These novel findings further confirm the importance of vitamin A in male fertility adding new insights into the retinoids complex biological framework. - Journal of Cellular Physiology, Volume 233, Issue 12, Page 9526-9537, December 2018.