One of the obstacles in irradiation therapy is cytoresistance, acquired by activation of self-defense systems, such as antioxidant or molecular chaperone systems, to cope with stress. We investigated whether irradiation preconditioning (IP) rendered resistance of the kidney against subsequent ischemia-reperfusion (I/R) and attempted to elucidate any such protective mechanisms. Mice were irradiated with a total of 4, 6, or 8 Gy using a cesium-137 source irradiator and then, 6 days later, were subjected to 28 min of bilateral renal ischemia followed by reperfusion. Eight Gy of IP significantly attenuated the increases in plasma creatinine (PCr) and blood urea nitrogen (BUN) concentration, structural damage, lipid peroxidation, superoxide formation, expression and activity of NADPH oxidase (NOX)-2, nitrotyrosine level, and hydrogen peroxide production after I/R in kidney tissues, indicating that IP protects the kidneys from I/R injury. IP markedly increased the activity of NOX, resulting in increased superoxide formation, manganese superoxide dismutase (MnSOD) activity and expression, and heat shock protein (HSP)-27 expression in kidneys. However, it did not change expressions of catalase, copper-zinc superoxide dismutase (CuZnSOD), and HSP-72. To investigate whether the protection afforded by IP was associated with increases in MnSOD and HSP-27 expression triggered by increased superoxide formation after IP, we administered manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin, a superoxide scavenger, to IP mice. This administration blocked superoxide formation and subsequent increases in MnSOD and HSP-27 expression and accelerated the post-I/R increases in PCr and BUN. In conclusion, IP renders kidney resistance to I/R injury, and this resistance is mediated by increased superoxide formation, which activates MnSOD activity and expression as well as HSP-27 expression.