In the endothelial cells, the nuclear factor-kappa B is an important intracellular signaling molecule by which changes in wall shear stress are transduced into the nucleus to initiate downstream endothelial nitric oxide synthase (NOS3) gene expression. We investigated whether the NFKB1 promoter polymorphism (^-94NFKB1 I/D) was associated with (1) NOS3 gene expression in endothelial cells under physiological levels of unidirectional laminar shear stress (LSS) and (2) endothelial function in Pre- and Stage I hypertensive individuals before and after a six-month supervised endurance exercise intervention. Competitive EMSAs revealed that proteins present in the nuclei of endothelial cells preferentially bound to the I allele NFKB1 promoter compared to the D allele. Reporter gene assays showed that the I allele promoter had significantly higher activity than the D allele. In agreement with these observations, the II genotype cells had higher p50 expression levels than the DD genotype cells. Cells with the homozygous insertional (II) genotype showed a greater increase in NOS-3 protein expression than did the deletional (DD) genotype cells under the LSS. Functional studies on volunteers confirmed higher baseline reactive hyperemic forearm blood flow, and further, the subgroup analysis revealed that DD homozygotes were significantly less prevalent in the responder group compared to the II and ID genotypes. We conclude that the ^-94NFKB1 I/D promoter variation contributes to the modulation of vascular function and adaptability to exercise-induced flow shear stress, most likely due to differences in NFKB1 gene transactivity.