To investigate the cardioprotective effect of methanolic extract of Gardenia gummifera root (MEGG) on Isoproterenol (ISO) induced myocardial infarction (MI) in rats. Methods: LCMS analysis of MEGG was done for the identification of cardioprotective constituents. Myocardial infarction was induced by the subcutaneous injection of ISO (6mg/100g body weight) at an interval of 24 h for 2 days. MEGG (125 and 250 mg/kg, p.o) was given to rats once daily for 45 days before the ISO challenge. The myocardial damage was assessed by quantifying the serum levels of cardiac marker enzymes (LDH, AST, ALT, CK-MB), serum iron and iron binding capacity, uric acid, and ceruloplasmin. Antioxidants such as catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST) and reduced glutathione (GSH) were altered in MI rats. The level of lipid peroxidation was measured as malondialdehyde (MDA), triphenyl tetrazolium chloride (TTC) test, and quantification of histopathological changes also supported the dose-dependent protective effects of MEGG. Results: MEGG significantly (p ≤ 0.05) protected the above-mentioned parameters from falling from normal levels. LCMS analysis of MEGG revealed the presence of cardioprotective constituents such as erythrodiol, lupeol, epicatechin, βsitosterol, asiatic acid, myricetin, oleanolic aldehyde, vernolic acid, chlorogenic acid, and dicaffeoylquinic acid. Conclusion: Our results suggest that MEGG affords a dose dependant cardioprotection against isoproterenol-induced myocardial infarction.