The objective of the present study was to investigate the effects of genistein and equol on 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3) in human and rat testis microsomes. These enzymes (3β-HSD and 17β-HSD3), along with two others (cytochrome P450 side-chain cleavage enzyme and cytochrome P450 17α-hydroxylase/17-20 lyase), catalyze the reactions that convert the steroid cholesterol into the sex hormone testosterone. Genistein inhibited 3β-HSD activity (0.2 μmol L− 1 pregnenolone) with half-maximal inhibition or a half-maximal inhibitory concentration (IC 50) of 87±15 (human) and 636±155 nmol L− 1 (rat). Genistein's mode of action on 3β-HSD activity was competitive for the substrate pregnenolonrge and noncompetitive for the cofactor NAD+. There was no difference in genistein's potency of 3β-HSD inhibition between intact rat Leydig cells and testis microsomes. In contrast to its potent inhibition of 3β-HSD, genistein had lesser effects on human and rat 17β-HSD3 (0.1 μmol L− 1 androstenedione), with an IC 50≥ 100 μmol L− 1. On the other hand, equol only inhibited human 3β-HSD by 42%, and had no effect on 3β-HSD and 17β-HSD3 in rat tissues. These observations imply that the ability of soy isoflavones to regulate androgen biosynthesis in Leydig cells is due in part to action on Leydig cell 3β-HSD activity. Given the increasing intake of soy-based food products and their potential effect on blood androgen levels, these findings are greatly relevant to public health.