Background: Methotrexate (MTX) is a chemotherapeutic drug, used mainly in many cancerous stages, inflammatory and auto-immune diseases, but its use has been limited by its nephrotoxicity. Cyanocobalamin is a water-soluble vitamin possessing nephro-protective properties. The aim of this study was to investigate the effect of cyanocobalamin on the nephrotoxicity of methotrexate. Methods: In the study 42 albino adult female rats were used, divided into six groups each containing seven rats (n=7). 1 st group: Control group (Negative control), 7 rats were injected intraperitoneally with 0.5ml/kg/day NS. Second group: 7 rats were injected intraperitoneally with a single dose of methotrexate (20 mg/kg) for 4 days. Third Group: 7 rats were given intraperitoneally cyanocobalamin at a dose (1.5 mg/kg/day) for two weeks, fourth, fifth, sixth group: 7 rats from each group were injected intraperitoneally with different concentrations of cyanocobalamin (0.5, 1, 1.5 mg/kg /day), respectively, for two weeks and MTX (20 mg/kg), which was injected only on day 11. On day 15, rats from all groups were euthanized, and blood samples were taken for biochemical tests, including evaluating serum urea and creatinine. The kidneys were extracted for histological investigation and evaluation of antioxidant (GSH) and oxidative stress (MDA) by using kidney tissue homogenates. Results: This study revealed that kidney damage, produced by the MTX (group II), is manifested by significantly elevated (P<0.05) urea and creatinine. On the contrary, the cyanocobalamin groups (IV, V, VI) significantly (P<0.05) reduced urea and creatinine. Renal antioxidant defense systems, such as reduced glutathione depleted by MTX therapy, were restored to normal levels by cyanocobalamin. Furthermore, cyanocobalamin reduced oxidative stress (MDA) and histologically reduced renal tissue injury induced by MTX. Conclusions: In conclusion, the study revealed that cyanocobalamin has a nephroprotective action upon MTX-induced renal damage in rats; cyanocobalamin may offer a protective effect, such as antioxidant action.