The hypoxia-reoxygenation (H/R) model helps analyze myocardial infarction triggered by acute myocardial ischemia, which induces cardiomyocyte proliferation and apoptosis. The Gene Expression Omnibus database was used to obtain the GSE74205 and GSE3866 microarray data, including microRNA (miRNA) and messenger RNA profiles, to catalog potential key miRNAs and genes. The role of rno-mir-496 expression in cardiomyocyte proliferation within 10 days of birth was established. The microRNA Target Prediction Database (miRDB) database-via Gene Ontology annotation-predicted hook microtubule tethering protein 3 (Hook3), a key target gene of rno-mir-496, was closely related to cell proliferation. Upregulation of miR-496 related to a significant reduction in apoptosis of H9c2 and human cardiomyocytes treatment with H/R. Moreover, transfection of H9c2 cells with miR-496 mimics, which were pretreated with H/R for 12 hours, increased Ki67 levels, proliferating cell nuclear antigen and Bcl-2 proteins; and decreased cleaved caspase-3 and Bax protein levels, as determined by reverse transcription-polymerase chain reaction and Western blot assays. A dual-luciferase reporter system confirmed that miR-496 targets the Hook3 suppressor. Hook3 overexpression stimulated apoptosis in H/R-treated cells, thus reducing cell proliferation. Upregulated miR-496 activated phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling, while Hook3 exhibited the inverse trend in H/R-treated H9c2 cells. In summary, with Hook3 functionality's aid, miR-496 upregulation defends cells from H/R-induced apoptosis and stimulates cell proliferation. miR-496 targets Hook3 to trigger the PI3K/Akt/mTOR signaling pathway for antiapoptotic and proliferative effects.