In enzymology, a columbamine oxidase (EC 1.21.3.2) is an enzyme that catalyzes the chemical reaction

2 columbamine + O₂ ${\displaystyle \rightleftharpoons }$ 2 berberine + 2 H₂O

Thus, the two substrates of this enzyme are columbamine and O₂, whereas its two products are berberine and H₂O.

This enzyme belongs to the family of oxidoreductases, specifically those acting on X-H and Y-H to form an X-Y bond with oxygen as acceptor. The systematic name of this enzyme class is columbamine:oxygen oxidoreductase (cyclizing). This enzyme is also called berberine synthase. This enzyme participates in alkaloid biosynthesis i. It employs one cofactor, iron.