Aim To investigate the nucleolar changes and the effect of heat shock protein 70 (HSP70) during the oxidative stress using an injury model of neonatal rat cardiomyocyte induced by hydrogen peroxide (H 2O 2). Methods The nucleolar impairment induced by 0.5 mmol/L H 2O 2 was observed with toluidine blue staining and electronic microscopy. The protective effect of HSP70 on nucleolar damage was investigated with heat shock pretreatment and antisense oligonucleotides of HSP70. Results H 2O 2 (0.5 mmol/L) significantly increased numbers of stained nucleolar granules, and resulted in obvious segregation of nucleolar components as indicated by staining with toluidine blue and electronic microscopy. Western blot analysis demonstrated that heat shock pretreatment induced expression of HSP70. Heat shock pretreatment significantly relieved the nucleolar impairment induced by H 2O 2. Moreover, it was showed by immunocytochemistry that H 2O 2 mediated HSP70 translocation from cytoplasm to nucleus and nucleolus in cardiomyocyte. Additional investigation showed that HSP70 antisense oligonucleotide can remarkably block the protective effect of HSP70 against the nucleolar injury induced by H 2O 2. Conclusions The nucleolar impairment of primary cultured cardiomyocyte can be induced by H 2O 2 and be protected by HSP70, which is over expressed and translocated to the nucleolus in the process.