Acute kidney injury (AKI) is characterized by mitochondrial dysfunction and activation of the innate immune system. The cyclic GMP-AMP synthase (cGAS) stimulator of interferon genes (STING) pathway detects cytosolic DNA and induces innate immunity. Here, we investigate the role of mitochondrial damage and subsequent activation of the cGAS-STING pathway using a genetically engineered animal model of cisplatin-induced AKI and cultured tubular cells. Cisplatin induced mtDNA leakage into the cytosol—probably through BCL-2-like protein 4 (BAX) pores in the mitochondrial outer membrane—in tubules, with subsequent activation of the cGAS-STING pathway, thereby triggering inflammation and AKI progression, which is improved in STING-deficient mice. STING knockdown in cultured tubular cells ameliorates inflammatory responses induced by cisplatin. mtDNA depletion and repletion studies support tubular inflammatory responses via the cGAS-STING signal activation by cytosolic mtDNA. Therefore, we conclude that mitochondrial dysfunction and subsequent activation of the mtDNA-cGAS-STING pathway is a critical regulator of kidney injury.