Objective Methotrexate (MTX) is an antimetabolite drug commonly prescribed for the various cancers and autoimmune diseases. Despite its considerable therapeutic effects, nephrotoxicity is the most important side-effect of treatment with MTX. Aquaporin1 (AQP1) is a water channel proteins which is present in mammalian kidney. Raspberry fruit with antioxidant properties is able to protect biological systems from the harmful effects of free radicals. The purpose of this study was to investigate the effect of raspberry extract on expression of AQP1 and the MTX-induced nephrotoxicity in rats. Materials and Methods In this experimental study, 60 adult male Wistar rats were divided into nine groups including control, sham, MTX treated group [single dose of 20 mg/kg of body weight (BW) MTX at the third day], raspberry treated groups [intraperitoneal (I.P) injection of 100, 200, 400 mg/kg of BW raspberry extract for ten consecutive days], MTX and raspberry treated groups. At day 11, rats were sacrificed via chloroform inhalation and kidney tissues were fixed in formalin solution for histological and immunohistochemistry analysis. The serological assays for urea, creatinine, uric acid and interleukin-6 (IL-6) levels were also performed. Results MTX elevated serum level of the urea, creatinine, uric acid, IL-6, renal tissue damage and decreased the AQP1 expression level. Raspberry fruit extract improved the kidney function and reduced side effects of MTX in treated rats. Expression of AQP1, in a dose dependent manner was also ameliorated, as compared to control group. Conclusion According to the findings of this study, it can be concluded that biological activity of compounds presented in raspberry fruit extract especially anthocyanins may have chemo-protective effect on kidney function and AQP1 expression in rats treated by MTX.