Aim To study the protective effect of trimetazidine (TMZ) on hypoxia jury in the rat cardiac myocytes. Methods Primary myocardial cells of rat were cultured in incubator at 37℃with 94%N₂₊₅%CO₂₊₁%O₂. Cell viability was detected by MTT. Apoptosis was detected by Hoechst staining. Mitochondrial membrane potential(MMP) was detected by TMRE. Mitochondrial energy metabolism was detected by Oxygraph-2k. Caspase-3, cytochrome C and respiratory chain complex enzymes were detected by Western blot. Results Hypoxia could induce apoptosis in the rat cardiac myocytes, decrease of MMP, cytochrome C release to cytosol. Furthermore, hypoxia could decrease state 3 respiration and respiratory control rate, increase state 4 respiration and decrease the expression of respiratory chain complex enzymes. Meanwhile, TMZ could play a protective effect against the apoptosis induced by hypoxia in the rat cardiac myocytes. It may be related to its protective role on MMP and respiratory chain complex enzymes. Conclusion TMZ could inhibit the apoptosis of rat cardiac myocytes induced by hypoxia via the mitochondrial pathway.