Testosterone biosynthesis from its precursor androstenedione is thought to be exclusively catalysed by the 17β-hydroxysteroid dehydrogenases-HSD17B3 in testes, and AKR1C3 in the ovary, adrenal and peripheral tissues. Here we show for the first time that the glucocorticoid activating enzyme 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1) can also catalyse the 17β-reduction of androstenedione to testosterone, using a combination of in vitro enzyme kinetic assays, mathematical modelling, and molecular docking analysis. Furthermore, we show that co-expression of HSD11B1 and AKR1C3 increases testosterone production several-fold compared to the rate observed with AKR1C3 only, and that HSD11B1 is likely to contribute significantly to testosterone production in peripheral tissues.