Nuclear medicine has been using radiopharmaceuticals for the diagnostic and therapeutic purposes of many diseases. Technetium-(99m) methoxyisobutylisonitrile (Tc-99m sestamibi) is a lypophilic complex that has a positive-loaded isonitril group. Aim of the study is to investigate whether Tc-99m sestamibi, which is one of the mostly used radiopharmaceuticals in nuclear medicine field, causes oxidative damage or not in rats' heart after an injection. A total of 16 male Sprague Dawley rats were randomly divided into two groups: group I: Tc-99m sestamibi group, Tc-99m sestamibi administered intravenously with the dose of 25MBq; group II: control group, one dose of isotonic sodium chloride was administered intravenous with the same volume as Tc-99m sestamibi group. Malondialdehyde (MDA) and total oxidant status (TOS) were used as markers of oxidative stress-induced heart impairment. Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and total antioxidant status (TAS) activities were studied to evaluate the changes in the antioxidant status. In the Tc-99m sestamibi group (group I), animals treated with Tc-99m sestamibi produced a significant decrease in the activities of antioxidant enzymes (SOD and CAT), while MDA level increased when compared with control group (group II) in myocardial tissue (p<0.05). On the other hand, the GSH-Px activities were significantly increased in the Tc-99m sestamibi-treated rats compared with the untreated rats (p<0.05). There was no significant difference in the TAS and TOS levels of plasma.