Allopregnanolone (ALLO) and androsterone (ADT) are naturally occurring 3-hydroxysteroids that act as positive allosteric regulators of GABA_A receptors. In addition, ADT also activates nuclear farnesoid X receptor and ALLO activates pregnane X receptor. At least with respect to GABA_A receptors, the biological activity of ALLO and ADT depends on the 3-hydroxyl group and is lost upon its conversion to either 3-ketosteroid or 3-hydroxyl epimer. Such strict structure-activity relationships suggest that the oxidation or epimerization of 3-hydroxysteroids may serve as physiologically relevant mechanisms for the control of the local concentrations of bioactive 3-hydroxysteroids. The exact enzymes responsible for the oxidation and epimerization of 3-hydroxysteroids in vivo have not yet been identified, but our previous studies showed that microsomal NAD⁺-dependent short-chain dehydrogenases/reductases (SDRs) with dual retinol/sterol dehydrogenase substrate specificity (RoDH-like group of SDRs) can oxidize and epimerize 3-hydroxysteroids in vitro. Here, we present the first evidence that microsomal NAD⁺-dependent 3-hydroxysteroid dehydrogenase/epimerase activities are widely distributed in human tissues with the highest activity levels found in liver and testis and lower levels in lung, spleen, brain, kidney and ovary. We demonstrate that RoDH-like SDRs contribute to the oxidation and epimerization of ALLO and ADT in living cells, and show that RoDH enzymes are expressed in tissues that have microsomal 3-hydroxysteroid dehydrogenase/epimerase activities. Together, these results provide further support for the role of RoDH-like SDRs in human metabolism of 3-hydroxysteroids and offer a new insight into the enzymology of ALLO and ADT inactivation.