<p>Melatonin is mainly synthesized and secreted by pineal gland, and plays multiple functions, including its regulating effects on reproductive processes. Sperm capacitation is necessary for fertilization, but the effect of melatonin on mouse sperm capacitation remains to be elucidated. We thus investigated the roles of melatonin on capacitation by culturing the sperms from mouse cauda epididymis in the medium with different doses of melatonin. The results showed that 10^–7&#xa0;mol/L melatonin significantly enhanced the sperm capacitation by increasing the sperm tyrosine phosphorylation level, percentage of the capacitated sperms and intracellular calcium concentration. In addition, our in vitro and in vivo results showed that melatonin enhanced the fertilizing capacity by increasing the percentage of oocyte cleavage and the number of the fetuses from receptive females which were mated with melatonin-treated males. Mechanically, melatonin activated cyclic adenosine monophosphate (cAMP)/p-Protein kinase A (p-PKA) pathway. Furthermore, the melatonin-induced tyrosine phosphorylation of sperms was decreased by treatment of MT1 or PKA inhibitor. And the same result was found in the sperms cultured in non-capacitating medium (without bicarbonate and bovine serum albumin). Therefore, all those results indicate that MT1/cAMP/PKA pathway is one of key regulatory factors in melatonin-mediated sperm capacitation. The results here are crucial for understanding the molecular mechanisms by which melatonin regulates sperm capacitation, and providing theoretical support for controlling sperm capacitation during artificial insemination procedures.</p> Graphical Abstract <p></p>