The presence of antiphospholipid antibodies (aPLAs) is associated with arterial or venous thrombosis and/or recurrent fetal loss. The proposed pathogenic mechanisms for aPLA effects include the inflammatory activation of monocytes and endothelial cells. Toll-like receptors (TLRs) are candidate signaling intermediates. The aim of this study was to investigate the relative contribution of TLR2 and TLR4 in cell activation by aPLAs. Of 32 patient-derived aPLAs, 19 induced an inflammatory activation of human monocytes and umbilical vein endothelial cells (HUVECs). In HUVECs, inflammatory responses to these aPLAs were increased by TNF pretreatment, which increases the expression of TLR2 but not TLR4. Anti-TLR2 but not anti-TLR4 antibodies reduced the aPLA-induced activation of monocytes and HUVECs. aPLAs activated TLR2-expressing human embryonic kidney 293 (HEK293) cells but not TLR4-expressing cells. Binding studies demonstrated an interaction between aPLAs and TLR2 but not TLR4. A role for CD14, a coreceptor for TLR2 and TLR4, can be inferred by observations that anti-CD14 antibodies reduced responses to aPLAs in monocytes, and that responses in HEK293 cells expressing TLR2 and CD14 were greater than in HEK293 cells expressing TLR2 alone. Our results demonstrate a role for TLR2 and CD14 in human endothelial cell and monocyte activation by aPLAs.