•Testosterone maintains BTB, meiosis, Sertoli–spermatid adhesion and spermiation.•Testosterone acts via classical and non-classical pathways.•Testosterone regulates PTM, Leydig, and Sertoli cells via the androgen receptor.•The combined regulation of many genes by testosterone supports spermatogenesis. Testosterone is essential for maintaining spermatogenesis and male fertility. However, the molecular mechanisms by which testosterone acts have not begun to be revealed until recently. With the advances obtained from the use of transgenic mice lacking or overexpressing the androgen receptor, the cell specific targets of testosterone action as well as the genes and signaling pathways that are regulated by testosterone are being identified. In this review, the critical steps of spermatogenesis that are regulated by testosterone are discussed as well as the intracellular signaling pathways by which testosterone acts. We also review the functional information that has been obtained from the knock out of the androgen receptor from specific cell types in the testis and the genes found to be regulated after altering testosterone levels or androgen receptor expression.