Adriamycin (ADR)-induced nephropathy in animals is an experimental analog of human focal segmental glomerulosclerosis, which presents as severe podocyte injury and massive proteinuria and has a poorly understood mechanism. The present study was designed to test the hypothesis that the peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α-mitochondria axis is involved in ADR-induced podocyte injury. Using MPC5 immortalized mouse podocytes, ADR dose dependently induced downregulation of nephrin and podocin, cell apoptosis, and mitochondrial dysfunction based on the increase in mitochondrial ROS production, decrease in mitochondrial DNA copy number, and reduction of mitochondrial membrane potential and ATP content. Moreover, ADR treatment also remarkably reduced the expression of PGC-1α, an important regulator of mitochondrial biogenesis and function, in podocytes. Strikingly, PGC-1α overexpression markedly attenuated mitochondrial dysfunction, the reduction of nephrin and podocin, and the apoptotic response in podocytes after ADR treatment. Moreover, downregulation of PGC-1α and mitochondria disruption in podocytes were also observed in rat kidneys with ADR administration, suggesting that the PGC-1α-mitochondria axis is relevant to in vivo ADR-induced podocyte damage. Taken together, these novel findings suggest that dysfunction of the PGC-1α-mitochondria axis is highly involved in ADR-induced podocyte injury. Targeting PGC-1α may be a novel strategy for the treatment of ADR nephropathy and human focal segmental glomerulosclerosis.