Rheumatoid arthritis (RA) is a chronic inflammatory joint disease. Vitamin D has been postulated to have a role in autoimmune diseases. The aim of this study was to answer, “whether treatment with vitamin D can improve RA patient outcome”. Forty female patients with rheumatoid arthritis with the age of 49.38±13.11 years, and forty healthy females as control with age of 45.14±12.63 years were included in the study. Patients were given a dose of 50,000 IU/week of vitamin D for two months. Blood samples were withdrawn from patients before and after treatment to study the effect of vitamin D on disease activity and its impact on plasma levels of TNF-α. Vitamin impact on the expression of activation and regulatory proteins CD28, CD25, CD120 and TNF receptor I (GITR) on helper CD4 and cytotoxic T cells (CD8) were assessed using flow cytometry. A highly significant difference was detected in total vitamin D in the RA group after treatment with vitamin D (P< 0.0001). Additionally, a significant difference was detected in vitamin D3 in the RA group after treatment with vitamin D compared to the control group (P< 0.0129). The results showed that untreated RA patients had a significant increase in serum TNF- α compared to healthy subjects. After treatment with vitamin D the TNF- α value was significantly decreased (P<0.0001). A significant increase was detected in P38 MAPK in leukocytes of RA patients that were treated with Phorbol 12-myristate 13-acetate (PMA). Administration of 1, 25(OH) vitamin D3 (1,25 D) significantly (P<0.0291) decreased the levels of p38 MAPK in an activated sample. A significant decrease was also detected in regulatory cytotoxic T cells (CD8+ CD28-) in RA group after treatment with vitamin D (P < 0.0337). On the other hand, there was a significant increase in CD8+ CD28+ in RA group after treatment with vitamin D (P<0.05). A significant decrease was detected in GITR+ CD8+ in RA group after treatment with vitamin D (P< 0.0085). A significant increase was detected in CD4+ CD25+ in RA group after treatment with vitamin D (P<0.05). A significant decrease was detected in CD4+ CD120+ in RA group after treatment with vitamin D (P<0.05). A significant decrease was detected in the diagnostic marker for RA after treatment with vitamin D (DAS-28 (P-value = 0.000), CRP (P<0.0008), ESR (P<0.0001), Anti-MCV (P< 0.0001), and rheumatoid factor (P< 0.02620)). In conclusion, the treatment of RA patients with vitamin D showed an improvement in the biochemical and immunological parameters that led to an improvement in physical movement. Vitamin D levels’ improvement may be attributed to the modulation of P38 MAPK.