To clarify the roles of nuclear localization signal (NLS) in inhibitory effect of HSP70 on H2O2-mediated nucleolar segregation, four eukaryotic vectors pcDNA3.1(-)-HSP70^WT (wild type), pcDNA3.1(-)-HSP70^ΔNLS (truncated mutant of HSP70 without NLS), pEGFP-N1-HSP70WT, and pEGFP-N1- HSP70^ΔNLS were constructed respectively. To mimic oxidative stress in vitro, final concentration of 1.0 mmol/L hydrogen peroxide (H²O²) was added into cultured C²C¹² myogenic cells. Toluidine blue staining showed predominantly compact, centrally localized nucleoli in intact control cells, but in H²O²-treated cells, an early onset of nucleolar segregation could be found after 3 h. The nucleolar segregation could be inhibited significantly in heat shock-pretreated cells and pcDNA3.1(-)-HSP70^WT-transfected cells. Fluorescence microscopy and immunoblotting for nucleolar protein showed that HSP70^WT was localized within cytoplasm in normal cells and redistributed to nucleus and nucleolus at 1 h after treatment with H^2O2. NLS deletion mutation of HSP70 (pcDNA3.1(-)-HSP70^ΔNLS) abolished H²O²-mediated translocation of HSP70 from cytoplasm to nucleus and nucleolus, and subsequently, its inhibitory effect on H²O²-mediated nucleolar impairment. It was suggested that wild type of HSP70 can attenuate significantly nucleolar segregation induced by oxidative stress. By mediating translocation of HSP70 from cytoplasm to nucleus and nucleolus, NLS played important role in the inhibitory effect of HSP70 against nucleolar impairment induced by oxidative stress.