Objective— ATP binding cassette transporter G1 (ABCG1) facilitates cholesterol efflux from macrophages to mature high-density lipoprotein particles. Whether genetic variation in ABCG1 affects risk of atherosclerosis in humans remains to be determined. Methods and Results— We resequenced the core promoter and coding regions of ABCG1 in 380 individuals from the general population. Next, we genotyped 10 237 individuals from the Copenhagen City Heart Study for the identified variants and determined the effect on lipid and lipoprotein levels and on risk of myocardial infarction (MI) and ischemic heart disease (IHD). g.−376C>T, g.-311T>A, and Ser630Leu predicted risk of MI in the Copenhagen City Heart Study, with hazard ratios of 2.2 (95% confidence interval: 1.2–4.3), 1.7 (1.0–2.9), and 7.5 (1.9–30), respectively. These results were confirmed for g.−376C>T in a case-control study comprising 4983 independently ascertained IHD cases and 7489 controls. Expression levels of ABCG1 mRNA were decreased by approximately 40% in g.−376C>T heterozygotes versus noncarriers (probability values: 0.005–0.009). Finally, in vitro specificity protein 1 (Sp1) bound specifically to a putative Sp1 binding site at position −382 to −373 in the ABCG1 promoter, and the presence of the −376 T allele reduced binding and transactivation of the promoter by Sp1. Conclusion— This is the first report of a functional variant in ABCG1 that associates with increased risk of MI and IHD in the general population.