ABSTRACTAcute myocardial infarction (AMI) is a leading cause of mortality among cardiovascular diseases, yet effective therapies for AMI are limited. Previous studies have suggested cardioprotective effects of columbianadin (CBN), but its specific role in AMI and the underlying mechanisms remain unclear. This study aims to investigate whether CBN influences AMI and to elucidate the underlying mechanisms. We conducted a network pharmacology analysis to investigate the relationship between CBN and AMI. The AMI model was established by ligating the left anterior descending (LAD) artery in C57BL/6J mice, which were subsequently administered CBN. Hypoxic H9c2 cells were utilized to evaluate the effects of CBN in vitro. Our study revealed that CBN treatment significantly reduced myocardial infarction in AMI mice. It enhanced mitochondrial function and suppressed autophagy flux in hypoxic H9c2 cells. Furthermore, CBN downregulated the expression of LC3, Beclin1, and Atg 5 genes and proteins. In response to CBN treatment, the phosphorylation levels of PI3K, Akt, and mTOR increased. Notably, RAPA attenuated the protective effect of CBN in enhancing the survival of hypoxic H9c2 cells and abolished its regulation of autophagy‐related proteins via the PI3K/Akt/mTOR signaling pathway. In conclusion, CBN reduces myocardial damage by suppressing autophagy via the PI3K/Akt/mTOR signaling pathway in AMI mice and hypoxic H9c2 cells.