The semen quality is a basic and the most important indicator of male reproductive health. Mammalian spermatozoa undergo a series of complex structural and functional changes in the epididymis to mature and achieve fertilization capacity. Sperm cell maturation is mediated by a complex physiological process, which is synergistically regulated by a large number of transcription factors, hormones and other signaling molecules. In recent years, there is increasing evidence supporting the notion that functional proteins and sRNA (small RNAs) in epididymosomes participate in sperm maturation and fertilization process. In this review, we summarize the biological roles of functional proteins and two major sRNAs (tRNAs and miRNAs) in sperm maturation in epididymosomes, and provide some theoretical guidance and new ideas for treatments of low fertility, infertility and other reproductive diseases in men.