Aim To investigate the regulatory effects of Genipin on mitochondrial pathway apoptosis induced by ischemia and its mechanisms. Methods H9c2 cells were cultured and grouped. The control group was cultured with DMEM without drugs under normal oxygen condition, the hypoxia group was cultured with DMEM without drugs under hypoxia condition, the Genipin group was treated with DMEM containing 2.5 μmol/L, 5.0 μmol/L, 10.0 μmol/L Genipin under hypoxia condition, the NC inhibitor group was transfected with NC inhibitor under normal oxygen condition, and the NC inhibitor+hypoxia group was transfected with NC inhibitor under hypoxic conditions, NC inhibitors+hypoxia+Genipin group was transfected with NC inhibitor and treated with DMEM containing 10.0 μmol/L Genipin under hypoxic conditions, miR-499 inhibitor+hypoxia+Genipin group was transfected with miR-499 inhibitor and treated with DMEM containing 10.0 μmol/L Genipin under hypoxic conditions. The differences of myocardial enzyme content, apoptotic rate, expression of apoptotic gene and miR-499 among groups were compared. Results Genipin group could decrease lactate dehydrogenase (LDH), creatine phosphate kinase (CK), creatine phosphate kinase isoenzymes (CK-MB) contents in cell culture medium, cell apoptotic rate, Bax and caspase-3 expression in cells, and increased the expression of Bcl-xL and miR-499 in cells in a dose-dependent manner. Transfection with miR-499 inhibitors could reverse 10.0 μmol/L Genipin reducing LDH, CK, CK-MB content in cell culture medium, apoptotic rate, Bax and caspase-3 expression and increasing Bcl-xL expression. Conclusion Genipin regulates mitochondrial apoptosis induced by ischemia and hypoxia via miR-499.