Lipids and mitochondrial oxidative stress plays a crucial role in the development of cardiovascular disease, and mitochondria compartment is presumed as the main source and susceptible target of intracellular Reactive Oxygen Species (ROS). The present study, an attempt has been made to evaluate the mitochondrial protection in isoproterenol (ISO)-induced myocardial-infarction (MI) in male Wistar rats. The rats were divided into four groups (n=6). Group I received 0.5% CMC treated as normal control group. Group II received isoproterenol (85 mg/kg body weight) intraperitoneal (i.p.) for two consecutive days (14th and 15th days). Group III received fenugreek (250 mg/kg b.wt) intragastric intubation for 15 days. Group IV rats received fenugreek as in Group III and additionally isoproterenol was given for two consecutive days (14th and 15th days).The isoproterenol-induced rats indicated increase in the level of TBARS and decreased in the activities of mitochondrial antioxidants in MI rats, decrease in the levels of mitochondrial phospholipids and increase the levels of mitochondrial cholesterol, free fatty acids (FFAs), triglycerides (TGs) and the activities of mitochondrial enzymes like Isocitrate dehydrogenase (ICDH), Succinate dehydrogenase (SDH), Malate dehydrogenase (MDH) and α-Ketoglutarate dehydrogenase (α-KGDH) were increased in isoproterenol-induced rats. On treatments with fenugreek at a daily dose of (250 mg/kg b.wt) showed significantly decrease in the levels of mitochondrial lipid peroxidation, raise in the mitochondrial antioxidant levels and also diminish in the mitochondrial enzymes. The present study exposed that fenugreek ameliorates the mitochondrial damage in isoproterenol induced myocardial infarction by maintaining lipid peroxidation metabolism due to its free radical scavenging, mitochondrial lipids, antioxidants and mitochondrial enzymes. Histopathological study was also in correlation with the biochemical parameters. Key notes: fenugreek, isoproterenol, mitochondrial enzymes, antioxidants.