The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) regulates cell death. We sought to determine whether DNA-PKcs played a role in the tubular damage that occurs during acute kidney injury (AKI) induced by LPS injection (to mimic sepsis), cisplatin administration, or renal ischemia/reperfusion injury. Although DNA-PKcs normally localizes to the nucleus, we detected cytoplasmic DNA-PKcs in mouse kidney tissues and urinary sediments of human patients with septic AKI. Increased cytoplasmic amounts of DNA-PKcs correlated with renal dysfunction. Tubule cell–specific DNA-PKcs deletion attenuated AKI-mediated tubular cell death and changes in the abundance of various proteins with mitochondrial functions or roles in apoptotic pathways. DNA-PKcs interacted with Fis1 and phosphorylated it at Thr 34 in its TQ motif, which increased the affinity of Fis1 for Drp1 and induced mitochondrial fragmentation. Knockin mice expressing a nonphosphorylatable T34A mutant exhibited improved renal function and histological features and reduced mitochondrial fragmentation upon induction of AKI. Phosphorylation of Thr 34 in Fis1 was detectable in urinary sediments of human patients with septic AKI and correlated with renal dysfunction. Our findings provide insight into the role of cytoplasmic DNA-PKcs and phosphorylated Fis1 in AKI development.