Inflammation plays a key role in atherogenesis, perhaps promoted by bacterial and viral products present within the artery wall. Vascular smooth muscle cells (VSMC) can express certain bacterially-responsive Toll-like receptors (TLRs), which promote a proinflammatory and proliferative VSMC phenotype when activated, but it is unknown whether virally-activated TLR can regulate VSMC phenotype. Here we tested the role in VSMC of TLR3, which is activated by double-stranded (dsRNA), a molecular signature of viruses. VSMC from multiple vessel types, including human coronary artery (HCoASMC) and mouse aorta (MAoSMC), expressed TLR3 constitutively, and HCoASMC were exquisitely sensitive to dsRNA-stimulated release of monocyte chemoattractant protein-1 (MCP-1) and interleukin-6. dsRNA-induced MCP-1 release was abolished by siRNA-mediated TLR3 knockdown in HCoASMC, and was absent in TLR3-/- MAoSMC, but was unimpaired in TLR2-/- and in TLR4 signaling-deficient MAoSMC. Exposure to dsRNA also activated ERK1/2 and NF-B in both human and murine SMC, but these effects were absent in SMC from TLR3-deficient mice, demonstrating a crucial role of TLR3 signaling. Double-stranded RNA also stimulated proliferation of HCoASMC, indicated by increased DNA synthesis, and induced persistent elevations in the intracellular levels of growth-promoting mediators including interleukin-1 (IL-1) and phospho-ERK1/2. We conclude that exposure of HCoASMC to dsRNA elicits dramatic TLR3-mediated proinflammatory and proproliferative phenotypic changes, responses that could potentially be triggered by viral infection of cells within the arterial wall.