Aim To explore the effect of heat shock pretreatment on hydrogen peroxide (H 2O 2)-induced apoptosis and the second mitochondria-derived activator of caspases (Samc) release from mitochondria in C₂C₁₂ myogenic cells. Methods After heat shock pretreatment (42℃ 1 h, recover 12 h), C₂C₁₂ myogenic cells were exposed to H 2O 2 (0.5 mmol/L)for 6 h, 12 h, 24 h,and 36 h respectively. The apoptotic morphological changes and percentage of apoptotic nuclei of C₂C₁₂ myogenic cells were analyzed. Caspase-3, Caspase-9 activities were assayed by caspase colorimetric assay kit and Western-blot. Inducible heat shock proteins were detected using Western-blot analysis. The release of Smac from mitochondria to cytoplasm was observed by Western-blot and immunofluorescence analysis. Results ①After treated with H 2O 2 (0.5 mmol/L), a marked increase of mitochondrial Smac release, activation of caspase-9, caspase-3 and apoptosis were observed respectively in C₂C₁₂ myogenic cells.②Heat shock pretreatment induced expression of HSP70, HSP90,αB-crystallin and inhibited H 2O 2-mediated Smac release from mitochondria, and inhibited the activation of caspase-9, caspase-3 and subsequent apoptosis. Conclusions Heat shock pretreatment could protect C₂C₁₂ myogenic cells against H 2O 2-induced apoptosis, and its mechanism might involve heat shock proteins expression and inhibition of Samc release from mitochondria.