Hyperglycemia-induced overproduction of mitochondrial reactive oxygen species has emerged as a major player in diabetic vascular complications. Mammalian translocase of inner mitochondrial membrane 44 (TIM44) was identified by upregulation in diabetic mouse kidneys. TIM44 functions as a membrane anchor of mitochondrial heat-shock protein 70 (mtHsp70) to TIM23 complex and is involved in the import of mitochondria-targeted preproteins into mitochondrial matrix. The process is dependent on inner membrane potential and ATP hydrolysis on ATPase domain of mitochondrial heat-shock protein 70. Hemagglutination virus of Japan-envelope vector that carries pcDNA3.1 plasmid that contains the full-length cDNA of TIM44 and control plasmid were injected weekly into the tail vein of uninephrectomized streptozotocin-induced diabetic CD-1 mice. The gene delivery alleviated proteinuria and renal hypertrophy at 8 wk after the injection, inhibited renal cell proliferation and apoptosis, and suppressed superoxide production. In vitro experiments, using human proximal tubular (HK2) cells, revealed that the gene delivery of TIM44 reversed high glucose-induced metabolic and cellular abnormalities such as enhanced reactive oxygen species production, increased ATP contents, alterations in inner membrane potential, increased cell proliferation, and apoptosis. Transfection with siRNA and expressing vector of TIM44 revealed that TIM44 facilitates import of antioxidative enzymes such as superoxide dismutase and glutathione peroxidase into mitochondria. The gene delivery of TIM44 therefore seems to be beneficial for the maintenance of mitochondrial function and is a novel therapeutic approach for diabetic nephropathy.