Ischemic heart disease (IHD) is a significant cause of cardiovascular diseases. MicroRNAs (miRNAs) have been thought to be critical regulators in the heart diseases. The present study was aimed to investigate the effect of miR-129-5p on the autophagy and apoptosis by targeting ATG14 as well as how miR-129-5p worked through the PI3K/AKT/mTOR signaling pathway in HO-induced H9c2 cells. H9c2 cells were induced by HO, after which the expression of miR-129-5p was decreased. Reverse transcription-quantitative polymerase chain reaction (qRT-PCR) was performed to detect the expression level of miR-129-5p in H9c2 cells. In addition, the expression of miR-129-5p and ATG14 were overexpressed or down-regulated after transfection. The transfection efficiency was verified by qRT-PCR. Cell viability, cell apoptosis, and the expression of autophagy and apoptosis-related proteins were determined by CCK-8, flow cytometry and western blotting, respectively. Furthermore, GFP fusion protein analysis was used to detect the expression level of LC3II which was related to autophagy. As a result, cell viability was decreased and cell autophagy was increased in HO-induced H9c2 cells. MiR-129-5p overexpression inhibited cell injury caused by HO in H9c2 cells which was certified by the increased cell viability and decreased cell autophagy and apoptosis. In addition, ATG14 was demonstrated to be a target of miR-129-5p which inhibited cell injury by down-regulation of ATG14. Moreover, phosphorylation of PI3K/AKT/mTOR pathway was activated by miR-129-5p overexpression or ATG14 inhibition to alleviate the autophagy and apoptosis in HO-induced H9c2 cells. In conclusion, this study indicated that miR-129-5p inhibited autophagy and apoptosis in HO-induced H9c2 cells partly by down-regulation of ATG14 through the activation of PI3K/AKT/mTOR pathway.