Background and Objectives: Vitamin D deficiency in viral infection associated with autoimmune diseases is well docu- mented. This study assessed the prevalence of JC virus in patients with rheumatoid arthritis (RA) and systemic lupus erythe- matosus (SLE), and its correlation with vitamin D level. Materials and Methods: Serum and urine samples were collected from 50 patients with RA and SLE. DNA was extracted and subjected to PCR test. Positive PCR products were sequenced, phylogenetic tree was constructed to determine the JC virus genotype. The patient’s vitamin D level was evaluated. Results: Of 50 patients, 19 (38%) were diagnosed as RA, and 31 (62%) were identified as SLE. JC virus DNA was detected in 17 (34%) patients’ urine samples including 5 (26.3%) RA and 12 (38.7%) SLE cases. JC virus DNA was detected 2 (4%) in patients’ serum samples (one RA. and one SLE). JC virus genotype 3A was dominant. Interestingly, the SLE patients with positive JC virus showed lowered vitamin D compared to patients with negative JC virus (P<0.005). Conclusion: Given the high rate of JC virus, DNA detection and susceptibility of patients for PML development, it is rec- ommended that detection of JC virus DNA and vitamin D level should be implemented for patients with RA/SLE prior to treatment.