BACKGROUND Multiple sclerosis (MS) is an autoimmune, neuro-inflammatory disease of central nervous system affecting physical, emotional, and cognitive aspects of patients. Association of vitamin D deficiency and MS has been shown in previous studies. The aim of this study was to evaluate serum vitamin D level in MS cases and their sex-matched healthy siblings (who are genetically near similar) and non-relative sex-matched healthy controls. METHODS A total of 135 subjects enrolled in this case-control study. Group one (n = 45) consisted of patients with established MS. Group two (n = 45) included sex-matched healthy siblings of the group one and group three participants (n = 45) were non-relative sex-matched healthy controls. Demographic data (age, sex), level of education, daily sun exposure duration, and month of birth gathered for all. Serum sample of all participants was collected for 25-hydroxy vitamin D measurement. RESULTS There was no significant difference between vitamin D level, sun exposure duration, education level, and season of birth in three evaluated groups. Mean vitamin D level was 8.2 ± 10.1 (nmol/l) in women and 13.3 ± 7 (nmol/l) in men (P = 0.001). There was a significant positive correlation between daily sun exposure duration and vitamin D level in whole participants (r = 0.28, P < 0.001) as well as in MS patients (r = 0.32, P = 0.030). Mean vitamin D level was significantly lower in participants who have born in spring and summer. CONCLUSIONS Vitamin D deficiency is high among Iranian population as well as MS patients.