Objective The purpose of this study was to explore whether miR-494-3p inhibits the occurrence of mitochondrial autophagy in cardiomyocytes by inhibiting the expression of PGC1-a and to supplement the theoretical basis for the role of autophagy in cardiac injury induced by hypoxia/reperfusion (H/R).Methods The expression of miR-494-3p was detected by RT-qPCR, and the expression of PGC1-a, autophagy-related proteins (LC3, Beclin 1), apoptosis-related proteins (Bax and Bcl-2), PINK1/Parkin signaling pathway-related proteins (PINK1, Parkin) and mitochondrial change-related proteins (Mfn1, Mfn2, OPA1) was detected by Western blotting. The changes in mitochondrial membrane potential were detected by JC-1 staining (Delta?m). The formation of autophagosomes was observed by transmission electron microscopy. Cell proliferation activity was detected by CCK-8, and cell apoptosis was detected by flow cytometry. A dual-luciferase gene reporter assay identified a targeted binding site between miR-494-3p and PGC1-a.Results The results showed that miR-494-3p and PGC1-a were differentially expressed in H/R cardiomyocytes; that is, the expression of miR-494-3p was downregulated, and the expression of PGC1-a was upregulated. In addition, mitochondrial autophagy occurred in H/R cardiomyocytes. That is, LC3-II/LC3-I, Beclin 1, PINK1, and Parkin expression was upregulated, Mfn1, Mfn2, and OPA1 expression was downregulated, and the mitochondrial membrane potential was decreased. The transfection of miR-494-3p mimic can significantly improve the cell proliferation activity of cardiomyocytes and inhibit the occurrence of cardiomyocyte apoptosis and autophagy, while the transfection of miR-494-3p inhibitor has the opposite result. After transfection of the miR-494-3p mimic, treatment with autophagy inhibitors and activators changed the effects of miR-494-3p on cardiomyocyte proliferation and apoptosis. At the same time, the overexpression of PGC1-a reversed the promoting effect of miR-494-3p on cardiomyocyte proliferation and the inhibitory effect on apoptosis and autophagy.Conclusion MiR-494-3p can target and negatively regulate the expression of PGC1-a to inhibit mitophagy in cardiomyocytes.