In enzymology, a 16alpha-hydroxysteroid dehydrogenase (EC 1.1.1.147) is an enzyme that catalyzes the chemical reaction

a 16alpha-hydroxysteroid + NAD(P)⁺ ${\displaystyle \rightleftharpoons }$ a 16-oxosteroid + NAD(P)H + H⁺

The 3 substrates of this enzyme are 16alpha-hydroxysteroid, NAD⁺, and NADP⁺, whereas its 4 products are 16-oxosteroid, NADH, NADPH, and H⁺.

This enzyme belongs to the family of oxidoreductases, specifically those acting on the CH-OH group of donor with NAD⁺ or NADP⁺ as acceptor. The systematic name of this enzyme class is 16alpha-hydroxysteroid:NAD(P)⁺ 16-oxidoreductase. This enzyme is also called 16alpha-hydroxy steroid dehydrogenase.

• Meigs RA, Ryan KJ (1966). "16-alpha-hydroxysteroid dehydrogenase of rat kidney. Purification, assay, and properties". J. Biol. Chem. 241 (17): 4011–5. PMID 4380686.