Tildipirosin is a 16-membered ring, tribasic and semi-synthetic macrolide antibiotic. The aim of this study was to determine the safety of tildipirosin following a single treatment at doses of 4 mg/kg and 8 mg/kg via subcutaneous injection. In the current research, tildipirosin (SC, single treatment) was applied at the doses of 4 and 8 mg/kg to the Normal Dose (ND) and High Dose (HD) group, respectively. Blood samples were gained before (0th d) and after treatments on 0.25, 0.5, 1, 3 and 21 d’s. Hemogram, biochemical and oxidative stress parameters were determined from blood samples. High dose tildipirosin increased cardiac damage parameters including creatine kinase-MB mass and partially troponin I within the first day, and high dose tildipirosin decreased statistically significant the thiobarbituric acid reactive substances levels on 1 and 3 d compared with ND group. Statistically significance fluctuations within reference ranges were observed in the biochemical and hemogram values. In conclusion, high dose tildipirosin may cause cardiotoxicity in sheep. However, cardiography, monitorization and histopathological evaluations should be done to determination of adverse effects of different doses of tildipirosin in sheep.