Methotrexate is an antifolate that is widely used in the treatment of rheumatic disorders and malignant tumors. The efficacy of methotrexate is often limited by severe side effects and toxic sequelae, where oxidative stress is noticeable. In the present study, the possible protective effect of β-glucan in methotrexate-induced toxicity was investigated. Following a single dose of methotrexate injection (20 mg/kg), either saline or β-glucan (50 mg/kg; orally) was administered for 5 days. After decapitation of the rats, trunk blood was obtained and the ileum, liver and kidney were removed to measure tissue malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) activity and collagen content, as well as histological examination. Methotrexate caused a significant decrease in GSH levels, while MDA levels, MPO activity and collagen content were increased in all the tissues (P < 0.05–0.001). On the other hand, administration of β-glucan following methotrexate abolished the depletion of GSH and inhibited the increases in MDA, MPO activity and collagen content, while the histological analysis revealed that β-glucan attenuated the tissue damage. Stimulation index, an indicator of oxidative burst in the neutrophils, was decreased by methotrexate (P < 0.001), while β-glucan abolished this effect. Furthermore, increased leukocyte apoptosis and cell death in methotrexate-treated animals were inhibited by β-glucan (P < 0.05). Thus, the findings of the present study suggest that β-glucan, through its antioxidant and immunoregulatory effects, may be of therapeutic value in alleviating the leukocyte apoptosis, oxidative tissue injury and thereby the intestinal and hepatorenal side effects of methotrexate treatment.