Capsaicin(Cap) is an active component of chili peppers that is extracted from capsicum plants. Recent studies have reported that Cap can ameliorate myocardial ischemia/reperfusion(I/R) injury. Mitochondria play an important role in pathways of apoptosis induced by myocardial I/R injury. However, the underlying mechanisms of Cap that improve mitochondrial function during I/R injury is not yet understood. The aim of this study was to evaluate whether Cap regained normal mitochondrial function in myocardial I/R injury, and to further explore the underlying mechanisms of action involved. In this study, an acute myocardial anoxia/reoxygenation(A/R) injury model was established using H9c2 cells. The cell viability was detected by MTS assay. LDH activity, mitochondrial permeability transition pores(mPTP) opening, and caspase-3 activity were analyzed using an ultra violet spectrophotometer. Levels of intracellular reactive oxygen species and apoptosis were evaluated by flow cytometry. Western blot analysis was used to determine the expression of 14-3-3η, Bcl-2, and Bax. The data showed that pretreatment with Cap decreased LDH release and increased cell viability in H9c2 cells that underwent A/R. Cap pretreatment significantly attenuated generation of reactive oxygen species, inhibited mPTP opening and caspase-3 activation, downregulated Bax, upregulated 14-3-3η and Bcl-2, and ultimately reduced apoptosis in H9c2 cells that underwent A/R. Moreover, 14-3-3ηRNAi adenovirus markedly eliminated the protective effects of Cap in H9c2 cells that underwent A/R. In addition, ABT-737(inhibitor of Bcl-2) significantly eliminated Cap protection. Taken together, the present study suggested that the cardioprotective effect of Cap against A/R injury involves the 14-3-3η pathway and prevention of mitochondrial damage.