Myocardial ischemia-reperfusion (I/R) injury, often arising from interventional therapy for acute myocardial infarction, leads to irreversible myocardial cell death. While previous studies indicate that nucleostemin (NS) is induced by myocardial I/R injury and mitigates myocardial cell apoptosis, the underlying mechanisms are poorly understood. Here, our study reveals that NS upregulation is critical for preventing cardiomyocyte death following myocardial I/R injury. Elevated NS protein levels were observed in myocardial I/R injury mouse and rat models, as well as Hypoxia/reoxygenation (H/R) cardiac cell lines (H9C2 cells). We identified binding sites for c-Jun and HIF-1α in the NS promoter region. Inhibition of JNK and HIF-1α led to a significant decrease in NS transcription and protein expression. Furthermore, inhibition of autophagy and NS expression promoted myocardial cell apoptosis in H/R. Notably, the cell model showed reduced LC3I transformation to LC3II, downregulated Beclin1, upregulated p62, and altered expression of autophagy-related proteins upon NS interference in H/R cells. These findings suggest that NS expression, driven by c-Jun and HIF-1α pathways, facilitates autophagy, providing protection against both myocardial I/R injury and H/R-induced cardiomyocyte apoptosis.