Objective:To investigate the role of oxidative stress in human renal tubular epithelial cells (HK-2) induced by high glucose and the underlying signal pathway in vitro. Methods:MYPT1, pro-caspase-3, PGC-1α , and Drp1 protein expressions were measured by West blot. MnSOD2, Drp1 and PGC-1α mRNA expressions were detected by real time PCR. Results:Results showed that high glucose significantly up-regulated the protein expressions of MYPT1, pro-caspase-3 and the mRNA expression of MnSOD2 in HK-2 cells; while Rho kinase inhibitor fasudil and ROCK1 siRNA inhibited protein expressions of pro-caspase-3 and the mRNA expression of MnSOD2 in HK-2 cells induced by high glucose. Importantly, fasudil and ROCK1 siRNA markedly inhibited the expressions of mitochondrial motor proteins Drp1 and mitochondrial gene PGC-1α in HK-2 cell=s induced by high glucose. Conclusions:Our findings suggest that Rho kinase signal pathway is involved in mitochondrial oxidative damage and apoptosis in high glucose-induced renal tubular epithelial cells by regulating mitochondrial motor proteins Drp1 and mitochondrial gene PGC-1α . Targeting Rho kinase signal pathway might be a potential strategy for the treatment of diabetic nephropathy.