The present study was designed to evaluate the cardioprotective effect of Tunisian flaxseed oil (Linum usitatissimum) against isoproterenol-induced myocardial infarction in rats by studying hypertensive and cardiac damage markers especially electrocardiographic changes and troponin T serum level. In vitro, the extracted oil showed an important inhibition of angiotensin converting enzyme (ACE) with an IC50 = 85.96 μg/ml. According to chemical analysis, this extract is composed essentially of alpha linolenic acid (ALA), an n-3 polyunsaturated fatty acid (58.59 %). Male rats were randomly divided into three groups, namely control (C), isoproterenol (ISO), and isoproterenol-treated group with flaxseed oil (FO + ISO). Isoproterenol injection showed changes in ECG pattern, including ST-segment elevation (diagnostic of myocardial infarction), increase in the serum levels of Troponin T and cardiac injury markers (creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), aspartate transaminase (AST), and alanine transaminase (ALT)). However, Linum oil pre-co-treatment prevented almost all the parameters isoproterenol-induced myocardial infarction in rats. Results of the present study proved that flaxseed oil has a significant effect by heart protection against isoproterenol-induced myocardial infarction through beneficial effect of the important fraction of ALA.