HSD17B3 is a testicular enzyme that catalyzes the final step in testosterone biosynthesis, converting the inactive androgen androstenedione to testosterone 1. The enzyme catalyzes the conversion of 17-oxosteroids to 17β-hydroxysteroids using NADPH as a cofactor, distinguishing it from related hydroxysteroid dehydrogenases that utilize NADH 1. HSD17B3 is highly expressed in fetal testes during early gonadal development, concurrent with other steroidogenic genes critical for male sexual differentiation 2. In humans, HSD17B3 deficiency causes 46,XY disorders of sex development characterized by ambiguous or female-appearing external genitalia despite internally normal testes and Wolffian structures 34. Patients with biallelic HSD17B3 mutations present with elevated androstenedione-to-testosterone ratios in blood, a diagnostic hallmark of deficiency 35. Notably, HSD17B3-deficient male mice maintain normal testosterone production and fertility, suggesting alternative compensatory enzymes—particularly HSD17B7 and HSD17B12—can partially compensate for HSD17B3 loss in mice but not humans 65. This species difference highlights the critical and non-redundant role of HSD17B3 in human testosterone production during prenatal development.