Human 12/15-lipoxygenase (ALOX15) catalyzes the oxidation of polyunsaturated fatty acids and has been implicated in the pathogenesis of atherosclerosis. We previously reported that a common haplotype of the ALOX15 gene is associated with higher prevalence of coronary artery calcification in a cohort of middle-aged African-Americans. This haplotype was uniquely tagged by a promoter variant (rs2255888). We carried out an in vitro characterization of this promoter variant to further investigate regulatory mechanisms of the ALOX15 gene. We evaluated the activity of ALOX15 variant A-carrying and wild type G-carrying promoter haplotypes using a luciferase assay. We demonstrated a 65% higher activity of the A-carrying promoter haplotype as compared to the G-carrying promoter haplotype. Using mass-spectrometry and electrophoretic mobility shift assay we showed that vimentin, a structural protein, specifically binds to both A-carrying and G-carrying promoter haplotypes in vitro. However, the A allele-carrying promoter haplotype had an 83% increased in vitro binding of vimentin compared to the G allele-carrying promoter haplotype. In vivo binding of vimentin to this particular promoter region has also been detected by chromatin immunoprecipitation. Using non-denaturing polyacrylamide gel electrophoresis and UV thermal melting we showed evidence of the formation of intramolecular G-quadruplex structure in the DNA sequence of the G-carrying promoter haplotype. However, for the A-carrying promoter haplotype, formation of intramolecular G-quadruplex structure was reduced and intermolecular G-quadruplex was increased. We also observed that vimentin increased ALOX15 gene expression. The current studies indicate that sequence variant of the rs2255888 polymorphism is associated with a structural change of ALOX15 promoter region. This change may be responsible for the increased binding of vimentin to the promoter region. Our study is the first to show that vimentin may act as a transcription regulator of ALOX15. It has been reported that vimentin is secreted by activated macrophages. Thus, the novel function identified here may be relevant to ALOX15 pathogenesis mechanisms in atherosclerosis.