Jasmonates (JAs) orchestrate immune responses upon wound/herbivore injury or infection by necrotrophic pathogens. Elucidation of catabolic routes has revealed new complexity in jasmonate metabolism. Two integrated pathways attenuate signaling by turning over the active hormone jasmonoyl-isoleucine (JA-Ile) through ω-oxidation or deconjugation, and define an indirect route forming the derivative 12OH-JA. Here, we provide evidence for a second 12OH-JA formation pathway by direct jasmonic acid (JA) oxidation. Three jasmonic acid oxidases (JAOs) of the 2-oxoglutarate dioxygenase family catalyze specific oxidation of JA to 12OH-JA, and their genes are induced by wounding or infection by the fungus Botrytis cinerea. JAO2 exhibits the highest basal expression, and its deficiency in jao2 mutants strongly enhanced antifungal resistance. The resistance phenotype resulted from constitutive expression of antimicrobial markers rather than from their higher induction in infected jao2 plants and could be reversed by ectopic expression of any of the three JAOs in jao2. Elevated defense in jao2 was dependent on the activity of JASMONATE RESPONSE 1 (JAR1) and CORONATINE-INSENSITIVE 1 (COI1) but was not correlated with enhanced JA-Ile accumulation. Instead, jao2 mutant lines displayed altered accumulation of several JA species in healthy and challenged plants, suggesting elevated metabolic flux through JA-Ile. Collectively, these data identify the missing enzymes hydroxylating JA and uncover an important metabolic diversion mechanism for repressing basal JA defense responses.