Hepatotoxicity is one of the most common side effects of methotrexate (MTX) therapy in patients. The aim of this study was to evaluate the protective effect of vitamin E against MTX-induced hepatotoxicity using 99m Tc-phytate as a radiopharmaceutical agent in animals. Rats were divided into five groups as follows: control, solvent, Vit E (100 mg/kg), MTX (20 mg/kg) and Vit E + MTX. Animals were intraperitoneally injected with Vit E for 17 days before MTX injection and continued for 4 days. Tc-phytate was injected into the tail of rats after the 21 days of Vit E administration. Percentage of the injected dose per gram of liver and spleen tissues (%ID/g) was calculated in treated rats. Liver imaging was obtained with gamma camera. In other experiment, liver of treated rats was assessed for histopathology. Tc-phytate uptake (%ID/g) of livers in control, solvent, Vit E, MTX and Vit E + MTX groups were 8.99% ± 1.37, 8.53% ± 2.91, 8.65% ± 3.84, 3.22% ± 1.09 and 8.38% ± 2.68. Vit E administration resulted in a significant increase of the level of %ID/g in MTX-injected animal. Vit E pre-treatment improved the shape of liver in MTX-treated rat which was seen abnormal view in planar imaging. Histophatological examinations approved the protective effect of Vit E against MTX-induced hepatotoxicity in rats. The results of this study show that Vit E significantly attenuates the MTX-induced hepatotoxicity in rats, and 99m Tc-phytate is an acceptable radiopharmaceutical agent for assessment of liver and spleen damages in the animal model.