The phytohormone salicylic acid (SA) plays essential roles in biotic and abiotic responses, plant development, and leaf senescence. 2,5-Dihydroxybenzoic acid (2,5-DHBA or gentisic acid) is one of the most commonly occurring aromatic acids in green plants and is assumed to be generated from SA, but the enzymes involved in its production remain obscure. DMR6 (Downy Mildew Resistant6; At5g24530) has been proven essential in plant immunity of Arabidopsis (Arabidopsis thaliana), but its biochemical properties are not well understood. Here, we report the discovery and functional characterization of DMR6 as a salicylic acid 5-hydroxylase (S5H) that catalyzes the formation of 2,5-DHBA by hydroxylating SA at the C5 position of its phenyl ring in Arabidopsis. S5H/DMR6 specifically converts SA to 2,5-DHBA in vitro and displays higher catalytic efficiency (K cat/K m = 4.96 × 10⁴ M⁻¹ s⁻¹) than the previously reported S3H (K cat/K m = 6.09 × 10³ M⁻¹ s⁻¹) for SA. Interestingly, S5H/DMR6 displays a substrate inhibition property that may enable automatic control of its enzyme activities. The s5h mutant and s5hs3h double mutant overaccumulate SA and display phenotypes such as a smaller growth size, early senescence, and a loss of susceptibility to Pseudomonas syringae pv tomato DC3000. S5H/DMR6 is sensitively induced by SA/pathogen treatment and is expressed widely from young seedlings to senescing plants, whereas S3H is more specifically expressed at themature and senescing stages. Collectively, our results disclose the identity of the enzyme required for 2,5-DHBA formation and reveal a mechanism by which plants fine-tune SA homeostasis by mediating SA 5-hydroxylation.