Mast cells are involved in both the genesis of allergic inflammation and in host defense; and reside in tissues where their location and responsiveness is regulated in part by adhesion to extracellular matrix proteins (ECM). We have reported that human mast cells (huMC) express TLR1-7, and 9 and respond to toll-like receptors (TLR) ligands by releasing cytokines and leukotriene C4. To determine if TLR ligation could similarly affect mast cells via an influence on adhesion, we employed huMC; and as substrates, fibronectin (FN) and vitronectin (VN). huMC were thus treated with double-stranded RNA (dsRNA) and adhesion to ECM was quantified. FcvarepsilonRI dependent mast cell degranulation was assessed. Adhesion molecule expression and activation was measured by flow cytometry. Activation of huMC through TLR3 with increasing amounts of polyI:C inhibited mast cell adhesion in a dose-dependent manner. This decrease in adhesion was accompanied by a similar decrease in IgE-mediated mast cell degranulation. Activation of TLR3 on huMC resulted in a change in the conformation of CD29, the receptor for FN, to an inactive form. Thus, TLR3 activation decreases mast cell attachment to VN and FN through an active process and one, which would abrogate mast cell attachment dependent potentiation of IgE-mediated responses.