The genetic mechanisms underlying the susceptibility to acute respiratory distress syndrome (ARDS) are poorly understood. We previously demonstrated that sphingosine 1-phosphate (S1P) and the S1P receptor, S1PR3, are intimately involved in lung inflammatory responses and vascular barrier regulation. Furthermore, plasma S1PR3 protein levels were shown to serve as a biomarker of severity in critically ill ARDS patients. This study explores the contribution of single nucleotide polymorphisms (SNPs) of the S1PR3 gene to sepsis-associated ARDS. S1PR3 SNPs were identified by sequencing the entire gene and tagging SNPs selected for case-control association analysis in African and European descent samples from Chicago, with independent replication in a European case-control study of Spanish individuals. Electrophoretic mobility shift assays, luciferase activity assays, and protein immunoassays were utilized to assess the functionality of associated SNPs. A total of 80 variants, including 29 novel SNPs, were identified. Due to limited sample size, conclusive findings could not be drawn in African descent ARDS subjects, however significant associations were found for two promoter SNPs (rs7022797 -1899T/G; rs11137480 -1785G/C), across two European descent samples supporting the association of alleles -1899G and -1785C with decreased risk for sepsis-associated ARDS. In addition, these alleles significantly reduced transcription factor binding to the S1PR3 promoter, reduced S1PR3 promoter activity, a response particularly striking after TNF-α challenge, and were associated with lower plasma S1PR3 protein levels in ARDS patients. These highly functional studies support S1PR3 as a novel ARDS candidate gene and a potential target for individualized therapy.