Because changes in subendothelial matrix composition are associated with alterations of the endothelial immune phenotype, we sought to understand if cytokine-induced NF-κB activity and downstream effects depend on substrate adherence of endothelial cells (EC). We compared the upstream phosphorylation cascade, activation of NF-κB, and expression/secretion of downstream effects of EC grown on tissue culture polystyrene plates (TCPS) with EC embedded within collagen-based matrices (MEEC). Adhesion of natural killer (NK) cells was quantified in vitro and in vivo. NF-κB subunit p65 nuclear levels were significantly lower and p50 significantly higher in cytokine-stimulated MEEC than in EC-TCPS. Despite similar surface expression of TNF-α receptors, MEEC had significantly decreased secretion and expression of IL-6, IL-8, MCP-1, VCAM-1, and ICAM-1. Attenuated fractalkine expression and secretion in MEEC (two to threefold lower than in EC-TCPS; p < 0.0002) correlated with 3.7-fold lower NK cell adhesion to EC (6,335 ± 420 vs. 1,735 ± 135 cpm; p < 0.0002). Furthermore, NK cell infiltration into sites of EC implantation in vivo was significantly reduced when EC were embedded within matrix. Matrix embedding enables control of EC substratum interaction. This in turn regulates chemokine and surface molecule expression and secretion, in particular of those compounds within NF-κB pathways, chemoattraction of NK cells, local inflammation, and tissue repair.