Fertilization is a stepwise process that allows two mature gametes to reach each other, fuse and eventually give rise to a new individual. Despite the tremendous importance of reproduction for species development and maintenance, fertility is decreasing worldwide, with peaks in western countries. It is estimated that about 7% of men experiences problems in conceiving a child because of sperm defects. In such a situation, understanding which are the essential sperm players in each of the steps of the fertilization process is essential for the development of new pharmacological strategies to treat the infertile men, for genetic screening of idiopathic male infertility as well as to produce effective male contraceptive agents. The present review will summarize recent evidence for the identification and characterization of molecular markers of sperm functions with emphasis on post-ejaculatory maturation events and the process of sperm-oocyte interaction.