Cellular localization determines whether the serine protease HtrA2 exerts pro- or antiapoptotic functions. In contrast to the well-characterized proapoptotic function of cytosolic HtrA2, mechanisms underlying the mitochondrial protective role are poorly understood. Mpv17l is a transmembrane protein previously implicated in peroxisomal reactive oxygen species metabolism and a close homolog of the inner mitochondrial membrane protein Mpv17. Here we demonstrate a previously undescribed direct interaction between Mpv17l and HtrA2 in mitochondria. The interaction is mediated by a PDZ domain and induces protease activation of HtrA2. HtrA2 inhibits mitochondrial superoxide generation, stabilizes mitochondrial membrane potential, and prevents apoptosis at baseline and in response to extracellular inducers of mitochondrial stress. The physiological role of Mpv17l is underscored by the finding that oxidative stress-induced downregulation of Mpv17l is a consistent feature in renal injury models. Our findings identify Mpv17l as a unique interacting protein and regulator of HtrA2 protease mediating antioxidant and antiapoptotic function in mitochondria.