Objective: To clone human cardiac heat shock protein 27(HSP27) gene and to determine the effects of HSP27 on the oxidative stress in rat cardiomyocyte cell line H9c2. Methods: Full length of HSP27 cDNA which got by RT-PCR was constructed into pCDNA3.1 + . The recombinant was transfected into rat cardiomyocyte cell line H9c2 and the stable transfection cell line was selected by G418. Then we observe the effects of HSP27 over-expression on LDH release and apoptosis induced H2O2 in H9c2. Results:1000 μmol/L H2O2 in HSP27 over-expression group and wild type group were 0.396 ± 0.017 vs. 0.390 ± 0.009 ( p ＞ 0.05 ), 0.437 ± 0.014 vs. 0.416 ± 0.015 ( P ＜ 0.05), 0.471 ± 0.018 vs. 0.417 ± 0.009 ( P ＜ 0.001), 0. 505 ± 0.030 vs. 0. 657 ± 0.022 ( P ＜ 0.001),and wild type group were (10.693 ± 1.122) ％ vs. (4.027 ± 1.628 )％ ( P ＜ 0.01). Conclusion: We cloned and constructed human cardiac HSP27 gene successfully, and over-expression of human HSP27 could inhibit oxidative damage significantly in H9c2.