This study was aimed to evaluate the preventive effect of rutin on lysosomal enzymes in isoproterenol induced cardio toxic rats. Male albino Wistar rats were pretreated with rutin (80mg/kg) daily for a period of 42days. After the pretreatment period, isoproterenol (100mg/kg) was subcutaneously injected to rats at an interval of 24h for two days. The activity of serum creatine kinase-MB and the levels of serum troponins T and I and the activities of serum and heart lysosomal enzymes (β-glucuronidase, β-N-acetylglucosaminidase, β-galactosidase, cathepsin-B and D) were increased significantly (P<0.05) in isoproterenol induced cardio toxic rats. Isoproterenol induced cardio toxic rats also resulted in decreased stability of membranes, which was reflected by decreased activities of β-glucuronidase and cathepsin-D in mitochondrial, nuclear, lysosomal and microsomal fractions. Pretreatment with rutin daily for a period of 42days to isoproterenol induced cardio toxic rats prevented the changes in the activities of these enzymes. Oral treatment with rutin (80mg/kg) to normal rats did not show any significant effect in all the biochemical parameters studied. Histopathology of myocardium showed the preventive effects of rutin in isoproterenol induced cardio toxic rats. In vitro study also confirmed the mechanism of action of rutin. Thus, the results of our study show that rutin protects the lysosomal membrane against isoproterenol induced cardiac damage. The observed effects are due to the free radical scavenging, antioxidant and membrane stabilizing properties of rutin.