Methotrexate (MTX), an antineoplastic drug, causes bone deformations. Arginine-silicate- inositol (ASI) complex is a combination of arginine, silicon and inositol that have been shown to have beneficial effects on bone health. In this study, it was investigated the potential protective role of ASI against MTX induced bone damage in rats. For this purpose, a total of 28 Sprague-Dawley male rats were divided into four groups: i) Control; ii) ASI, the rats were treated orally with 25 mg/kg/day of ASI for 15 days; iii) MTX, the rats were injected subcutaneously with 0.75 mg/kg of MTX on 8^th and 12^th days for 5 days, and iv) ASI+ MTX, the rats were treated orally with 25 mg/kg/day of ASI for 15 days and injected with MTX on 8^th and 12^th days for 5 days. MTX significantly increased aspartateaminotransferase (AST), alanineaminotransferase (ALT), urea (BUN) and creatine levels, while decreased tibia osteocalcin levels (P<0.001). In the ASI+MTX group; ALT, AST, BUN and creatinine levels were significantly decreased compared to the MTX group. MTX increased bone nuclear factor-kappa B (NF-κB), receptor activator of NF-κB ligand (RANKL), interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) levels and decreased the bone osteoprotegerin (OPG) and type1collagen levels (P<0.001). However, ASI administration reduced the levels of NF-κB, RANKL, IL-6 and TNF-α, while increased the levels of OPG and type1collagen levels (P<0.05). Similarly, the protective effect of ASI against MTX was confirmed by histological analysis. In conclusion, ASI application reduced the negative effects of MTX on bone damage in rats.