Methotrexate (MTX), which has been used in clinical practice for approximately 70 years, is still widely employed in the treatment of rheumatoid arthritis (RA), psoriasis, and cancer. Although MTX toxicity causes nephrotoxicity, hepatotoxicity, bone marrow suppression, pulmonary fibrosis, and gastrointestinal damage, previous studies have not addressed splenic toxicity. This is the first study to examine the effectiveness of infliximab (INF) against MTX-induced toxicity in splenic tissues via the regulation of CD3, CD68, and C200R. We investigated the effects of MTX on macrophages and T lymphocytes in the spleen at the molecular level and examined the protective potential of the tumor necrosis factor (TNF)-alpha antagonist INF against MTX toxicity. Three groups of rats were set up. Group 1 received saline solution only, group 2 a single dose of MTX (20 mg/kg), and group 3 INF (7 mg/kg) before administration of a single dose of MTX (20 mg/kg). All injections were given intraperitoneally. Spleen tissues were removed 5 days after MTX administration and evaluated for CD3, CD68, and CD200R using immunohistochemical staining. Finally, the mean numerical density of CD3+, CD68+, and CD200R+ cells was estimated by a histopathologist using StereoInvestigator 8. MTX increased the numerical densities of CD3+, CD68+, and CD200R+ cells (p < 0.05). We also observed that INF reduced the numerical densities of these cells following MTX administration (p < 0.05). INF may, therefore, be a promising candidate for the prevention of the deleterious effects on spleen tissue of MTX, used in the treatment of RA and cancer.