Background: Research reports that serum Vitamin D levels are deficient in AMI patients. Aim: To compare serum Vitamin D levels between AMI patients and healthy controls. Methods: Study design was Cross Sectional Comparative. The study was conducted at Department of Physiology & Cell Biology, University of Health Sciences, Lahore 2018 to 2019. Serum vitamin D levels were measured in 80 study subjects. Among 80 study subjects, 40 had AMI and 40 were healthy controls. Results: The serum Vitamin D levels less than 20ng/ml were considered deficient. Mann Whitney U test was used to compare the groups. The mean serum Vitamin D levels of the AMI patients were significantly lower (7±2.47ng/ml) as compared to healthy controls (17.17±4.8ng/ml) at (p=0.000). Conclusion: AMI patients have significantly lower serum Vit. D levels as compared to healthy individuals. Keywords: Acute myocardial infarction, Serum Vitamin D