Background: Rheumatoid arthritis (RA) is a chronic autoimmune disease marked by inflammation, pain, and joint damage. Vitamin D deficiency worsens inflammation and increases RA disease activity. Aim of the study: This study aimed to explore the relationship between Vitamin D deficiency and disease activity in rheumatoid arthritis patients. Methods: This cross-sectional study was conducted in the Department of Medicine, Mymensingh Medical College Hospital, Mymensingh, Bangladesh from January 2003 to December 2003. The study included 100 rheumatoid arthritis patients, divided into two groups- Group A: 50 patients with normal Vitamin D level and Group B: 50 patients with Vitamin D deficiency. Result: No significant difference was found between the groups in demographic characteristics. Group B exhibited significantly greater disease severity compared to Group A. The Disease Activity Score (DAS28) was higher in Group B (4.2 ± 1.3) than in Group A (2.8 ± 1.1), with a p-value < 0.001. CRP levels were also elevated in Group B (12.4 ± 3.5 mg/L vs. 5.3 ± 2.1 mg/L; p < 0.001). Group B had more swollen joints (6.2 vs. 3.5) and tender joints (7.3 vs. 4.1), with p < 0.001. ESR levels were higher in Group B (38.7 vs. 22.4 mm/h; p < 0.001). Negative correlations were found between Vitamin D levels and DAS28 (r = -0.48), CRP (r = -0.51), and ESR (r = -0.52). Conclusion: This study concludes that Vitamin D deficiency is significantly associated with increased disease activity, inflammation, and joint involvement in rheumatoid arthritis patients.