The protective effects of heat shock pretreatment against the injury induced by hydrogen peroxide(H2O2) were evaluated in neonatal rat cardiomyocytes. The results showed that heat shock pretreatment significantly reduced cell mortality rate, LDH release rate and increased total antioxidation of the cells. Western blot analysis demonstrated that heat shock pretreatment induced expression of alpha B-crystallin and immunoelectron microscopy demonstrated H2O2-mediated alpha B-crystallin translocation from cytoplasm to nucleus and microfilament in cardiomyocytes. The results suggest that the mechanism of protective effect of heat shock pretreatment might involve the induction and translocation of alpha B-crystallin which then protect cardiomyocytes against H2O2-induced injury by stabilizing cytoskeletal structure and increasing anti-oxidation capacity of the cells.