Long-chain acyl-CoA synthetase 1 (ACSL1) converts free fatty acids into acyl-CoAs. Mouse studies have revealed that ACSL1 channels acyl-CoAs to β-oxidation, thereby reducing glucose utilization, and is required for diabetes-accelerated atherosclerosis. The role of ACSL1 in humans is unknown. We therefore examined common variants in the human ACSL1 locus by genetic association studies for fasting glucose, diabetes status and preclinical atherosclerosis by using the MAGIC and DIAGRAM consortia, followed by analyses in participants from the Multi-Ethnic Study of Atherosclerosis (MESA), the Penn-T2D consortium, and a meta-analysis of subclinical atherosclerosis in African Americans, and finally expression quantitative trait locus analysis and identification of DNase I hypersensitive sites. The results show that three SNPs in ACSL1 (rs7681334, rs735949 and rs4862423) are associated with fasting glucose or diabetes status in these large (>200,000 subjects) data sets. Furthermore, rs4862423 is associated with subclinical atherosclerosis, and coincides with a DNase I hypersensitive site highly accessible in human heart. SNP rs735949 is in strong linkage equilibrium with rs745805 significantly associated with ACSL1 levels in skin, suggesting tissue-specific regulatory mechanisms. This study provides evidence in humans of ACSL1 SNPs associated with fasting glucose, diabetes and subclinical atherosclerosis, and suggests links among these traits and acyl-CoA synthesis.