Stroke is one of the most common neurological disorder and third most common cause of death in the world. Low vitamin D concentrations have been shown to predict risk of cardiovascular disease and all-cause of mortality. The aim of this study was to estimate serum vitamin D level in acute ischemic stroke patients. This comparative cross-sectional type of study was conducted in the Department of Neurology and Department of Medicine at Mymensingh Medical College and Hospital, Bangladesh from November 2017 to June 2019 with a total number of 100 study subjects. Total fifty patients with acute ischemic stroke were enrolled in Group A and another fifty age and sex matched volunteer subjects were enrolled in Group B with no prior history of stroke or transient ischemic attacks. Serum vitamin D levels, fasting plasma glucose and lipid profile were assessed in both groups and compared with each other. P value <0.05 was considered as significant in the study. Mean fasting blood sugar, serum fasting total cholesterol (TC), serum fasting triglycerides, serum fasting Low density lipoprotein (LDL) were significantly higher in Group A than Group B (p<0.05). Serum vitamin D level in Group A was 25.28±8.47ng/ml and in Group B was 30.90±5.80, (p=0.001). Insufficient vitamin D level was found in 52.0% of ischemic stroke patients and in 30% of healthy controls (p=0.0002). Vitamin D deficiency was found in 20.0% ischemic stroke patients and 10.0% in healthy controls. This study demonstrates a positive association between low serum vitamin D level and acute ischemic stroke. Further studies are required to determine whether vitamin D supplementation could improve functional outcome in patients with ischemic stroke.