In enzymology, a (S)-6-hydroxynicotine oxidase (EC 1.5.3.5) is an enzyme that catalyzes the chemical reaction

(S)-6-hydroxynicotine + H₂O + O₂ ${\displaystyle \rightleftharpoons }$ 1-(6-hydroxypyridin-3-yl)-4-(methylamino)butan-1-one + H₂O₂

The 3 substrates of this enzyme are (S)-6-hydroxynicotine, H₂O, and O₂, whereas its two products are 1-(6-hydroxypyridin-3-yl)-4-(methylamino)butan-1-one and H₂O₂.

This enzyme belongs to the family of oxidoreductases, specifically those acting on the CH-NH group of donors with oxygen as acceptor. The systematic name of this enzyme class is (S)-6-hydroxynicotine:oxygen oxidoreductase. Other names in common use include L-6-hydroxynicotine oxidase, 6-hydroxy-L-nicotine oxidase, and 6-hydroxy-L-nicotine:oxygen oxidoreductase. It employs one cofactor, FAD.

• Dai VD, Decker K, Sund H (1968). "Purification and properties of L-6-hydroxynicotine oxidase". Eur. J. Biochem. 4 (1): 95–102. doi:10.1111/j.1432-1033.1968.tb00177.x. PMID 5646150.
• Decker K, Bleeg H (1965). "Induction and purification of stereospecific nicotine oxidizing enzymes from Arthrobacter oxidans". Biochim. Biophys. Acta. 105 (2): 313–24. doi:10.1016/s0926-6593(65)80155-2. PMID 5849820.