Methotrexate (MTX) has been in use for the treatment of rheumatoid arthritis (RA), psoriasis, and cancer since 1948. Its toxic side effects on tissues and organs have been well documented but splenotoxicity has not been addressed. This study set out to investigate this issue by examining the effectiveness of anti-TNF alpha agents against MTX-induced toxicity in T lymphocytes and macrophages via the regulation of CD3, CD68, and CD200R. Twenty-four Sprague Dawley rats were allocated to three groups: control (received saline solution only), MTX (20 mg/kg of single-dose of MTX), and Ada + MTX (single dose of 10 mg/kg Adalimumab before MTX administration). The spleens were removed 5 days after MTX administration. The number of CD3+/mm3 cells for the control, MTX and Ada + MTX groups were, respectively, 2.69 +/- 0.86, 20.51 +/- 2.7, (p = 0.000) and 11.07 +/- 2.01 (p = 0.000). The number of CD68+ macrophages/mm3 in the control, MTX and Ada + MTX groups were, respectively, 8.62 +/- 1.08, 38.19 +/- 1.37 (p = 0.000), and 16.87 +/- 12.57 (p = 0.000). The number of macrophages that were CD200R+/mm3 in the control, MTX, and Ada + MTX groups were 3.33 +/- 1.66, 25.77 +/- 2.37 (p = 0.000), and 8.68 +/- 2.66 (p = 0.000), respectively. We also observed that Ada reduced the numerical densities of these cells following MTX administration (p < 0.05). Ada may, therefore, be a promising candidate for the prevention of the deleterious effects on T lymphocytes and macrophages of MTX-induced toxicity.