In this chapter, we describe the formation of estradiol by Leydig cells and its effects on spermatogenesis and male fertility. Based on important discoveries made over 30 years, we focus our attention on the final step of sex hormone production, known as aromatization, and present the current knowledge of the aromatase enzyme, its localization, and gene expression in males. Additionally, we provide a summary of significant findings concerning estrogen receptors (ERα, ERβ, and GPER) and the genomic (classical) as well as non-genomic (nonclassical) effects induced by estradiol. To illustrate the role of estrogens in males, we discuss clinical and experimental evidence from case reports and studies utilizing models such as the aromatase knockout (ArKO) mice, estrogen receptors’ knockout mice (ERαKO, ERβKO, and ERαβKO) and GPER knockout mice (GPERKO). Finally, we give an overview of the results of recent studies, both in vitro and in vivo, on the molecular effects of estradiol on spermatogenesis and fertility.