Background: Intestinal toxicity is a serious side effect in methotrexate (MTX) chemotherapy. Objective: To investigate the mechanisms by which the anticancer drug MTX-induced intestinal damage could be prevented by thymoquinone (TQ), an active ingredient of Nigella sativa. Materials and Methods: TQ was given orally for 10 days, and MTX toxicity was induced at the end of day 3 of the experiment, with or without TQ pretreatment. Results: MTX caused intestinal damage, represented by distortion in normal intestinal histological structure, with significant oxidative stress, exhibited as decrease in reduced glutathione concentration and catalase activity, along with significant increase in malondialdehyde level compared to control group. MTX also caused nitrosative stress evident by increased intestinal nitric oxide (NO) level, with up-regulation of inducible NO synthase expression shown in immunohistochemical staining. Furthermore, MTX caused inflammatory effects as evident by up-regulation of intestinal necrosis factor-kappa beta and cyclooxygenase-2 expressions, which were confirmed by increased intestinal tumor necrosis factor-alpha level via enzyme-linked immunosorbent assay. Moreover, MTX caused apoptotic effect, as it up-regulated intestinal caspase 3 expression. Concomitant TQ significantly reversed the MTX-induced intestinal toxic effects by reversing intestinal microscopic damage, as well as significantly improving oxidative/nitrosative stress, inflammatory and apoptotic markers tested compared to MTX alone. Conclusion: TQ may possess beneficial intestinal protective effects as an adjuvant co-drug against MTX intestinal toxicity during cancer chemotherapy. TQ protection is conferred via antioxidant, anti-nitrosative, anti-inflammatory, and anti-apoptotic mechanisms.