In order to explore the role of Smac/DIABLO in H₂O₂-induced apoptosis of C₂C₁₂ myogenic cells, Hoechst 33258 staining was used to examine cell morphological changes and to calculate percentage of apoptotic nuclei. DNA ladder pattern on agarose gel electrophoresis was used to observe DNA fragmentation. The release of Smac/DIABLO from mitochondria to cytoplasm was observed by Western blotting. Activities of Caspase-3, Caspase-9 were assayed with Caspase Colorimetric Assay Kit and Western blotting. Full length Smac/DIABLO gene was transiently transfected in C₂C₁₂ myogenic cells by lipofectamine and then protein levels of Smac/DIABLO were analysed by Western blotting. The results showed that: (1)H₂O₂ (0.5 mmol/L) resulted in a marked release of Smac/DIABLO from mitochondrial to cytoplasm 1 h after treatment, activation of Caspase-3, Caspase-9 4 h after treatment and specific morphological changes of apoptosis 24 h after treatment; (2)Smac/DIABLO overexpression significantly enhanced H₂O₂ induced apoptosis in C₂C₁₂ myogenic cells as shown by specific DNA ladder pattern in agarose gel electrophoresis, increase of percentage of apoptotic nuclei and marked activation of Caspase-3, Caspase-9. These data suggested that H₂O₂ could result in apoptosis of C₂C₁₂ myogenic cells，and that release of Smac/DIABLO from mitochondrial to cytoplasm and the subsequent activation of Caspase-9 and Caspase-3 played important roles in H₂O₂-induced apoptosis in C₂C₁₂ myogenic cells.