BACKGROUND AND PURPOSE: Vitamin D deficiency is common across all age groups and may contribute to cardiovascular diseases. Serum 25-hydroxyvitamin D deficiency causing ischemic stroke has been documented in recent reports. AIM: To investigate the association of serum 25-hydroxyvitamin D deficiency with ischemic stroke and subtypes. METHODS: We recruited 250 consecutive ischemic stroke patients and 250 age and sex matched controls attending the Department of Neurology, at Yashoda hospital, Hyderabad, India, from January 2011 to December 2012. All ischemic stroke patients underwent stroke subtyping. We measured 25-hydroxyvitamin D by chemiluminescence test, serum calcium, phosphorus, alkaline phosphatase, and C-reactive protein (CRP) in cases and controls. RESULTS: Out of 250 stroke patients, 190 (76%) were men and mean age was 58.4+/-11.1 years (age range-26-89 years). 25-hydroxyvitamin D deficiency was observed in 122 (48.8%) stroke patients and 79 (31.6%) controls (P=0.001). Among stroke patients, serum 25-hydroxyvitamin D deficiency was found in 54.9% (50/91) of patients with large artery atherosclerosis, 54% (20/37) in cardioembolic stroke, 44.4% (20/45) in small artery diseases, 42.8% (15/35) in stroke of other determined etiology and 40.4% (17/42) in stroke of un-determined etiology. Multiple logistic regression analysis showed an independent association of 25-hydroxyvitamin D deficiency with ischemic stroke (odds ratio: 1.6; 95% CI 1.2-2.8). The association was strongest with large artery atherosclerosis (odds ratio: 2.4; 95% CI 1.6-3.5) and cardioembolic stroke (odds ratio: 2.0; 95% CI 1.0-3.2). CONCLUSIONS: We found that 25-hydroxyvitamin D deficiency had an independent association with ischemic stroke. The association was established in large artery arthrosclerosis and cardioembolic stroke.