Apolipoprotein (apo)CIII predicts risk for coronary heart disease. We recently reported that apoCIII directly activates human monocytes. Recent evidence indicates that toll-like receptor (TLR)2 can contribute to atherogenesis through transduction of inflammatory signals. Here, we tested the hypothesis that apoCIII activates human monocytoid THP-1 cells through TLR2. ApoCIII induced the association of TLR2 with myeloid differentiation factor 88, activated nuclear factor (NF)-κB in THP-1 cells, and increased their adhesion to human umbilical vein endothelial cells (HUVECs). Anti-TLR2 blocking antibody, but not anti-TLR4 blocking antibody or isotype-matched IgG, inhibited these processes (P<0.05). ApoCIII bound with high affinity to human recombinant TLR2 protein and showed a significantly higher (P<0.05) and saturable binding to 293 cells overexpressing human TLR2 than to parental 293 cells with no endogenous TLR2. Overexpression of TLR2 in 293 cells augmented apoCIII-induced NF-κB activation and β1 integrin expression, processes inhibited by anti-apoCIII antibody as well as anti-TLR2 antibody. Exposure of peripheral blood monocytes isolated from C57BL/6 (wild-type) mice to apoCIII activated their NF-κB and increased their adhesiveness to HUVECs. In contrast, apoCIII did not activate monocytes from TLR2-deficient mice. Finally, intravenous administration to C57BL/6 mice of apoCIII-rich very-low-density lipoprotein (VLDL), but not of apoCIII-deficient VLDL, activated monocytes and increased their adhesiveness to HUVECs, processes attenuated by anti-TLR2 or anti-apoCIII antibody. ApoCIII-rich VLDL did not activate monocytes from TLR2-deficient mice. In conclusion, apoCIII activated monocytes at least partly through a TLR2-dependent pathway. The present study identifies a novel mechanism for proinflammatory and proatherogenic effects of apoCIII and a role for TLR2 in atherosclerosis induced by atherogenic lipoproteins.