To investigate the effects and mechanisms of heat shock protein 70（HSP70）on the hypoxia/reoxygenation(H/R) injury of cardiomyocytes during hypoxic postconditioning(HPC), H9C2 cardiomyocytes were divided into four groups : the control group（cultured for 10 h under normal oxygen）,the H/R group (hypoxia for 4 h and reoxygenation for 6 h), the HPC group (3 cycles of 5 min hypoxia/reoxygenation after hypoxia for 4 h, then reoxygenation for 6 h), the HPC+quercetin(Quer) group（50 μmol/L HSP70 inhibitor Quer was added 1 h before HPC）. Morphological changes of cardiomyocytes were examined by Hoechst 33242 staining, and cardiomyocytes apoptosis were examined by FACS. The effects of HPC on the expression of HSP70, TLR4, NF-κB p65, TNF-α, IL-1β, IL-6, B-cell lymphoma 2(Bcl-2), Bcl-2-associated X protein(Bax) and Caspase-3 were determined by Western blotting. The results showed that the levels of HSP70 and Bcl-2 in the HPC group were significantly higher than those in the H/R group（P<0.05）, and TLR4, NF-κB p65, TNF-α, IL-1β, IL-6, Bax, Caspase-3 levels were significantly lower than those in the H/R group（P<0.05）. Whereas the levels of HSP70 and Bcl-2 in the HPC+Quer group were significantly lower than those in the HPC group（P<0.05）, and TLR4, NF-κB p65, TNF-α, IL-1β, IL-6, Bax , Caspase-3 levels were significantly higher than those in the HPC group（P<0.05）.Thus, HPC inhibits the TLR4/NF-κB signaling pathway and alleviates the H/R-induced inflammatory injury of cardiomyocytes by upregulating HSP70 expression.