Our previous study demonstrated that Toll-like receptor 4 (TLR4) could act as a co-receptor with annexin A2 (ANX2) mediating anti-β2-glycoprotein I/β2-glycoprotein I (anti-β2GPI/β2GPI)-induced tissue factor (TF) expression in human acute monocytic leukemia cell line THP-1. In the current study, we further explored the roles of TLR4 and its adaptors, MyD88 and TRIF, in anti-β2GPI/β2GPI-induced the activation of human blood monocytes and THP-1 cells and the relationship among TLR4, β2GPI and ANX2 in this process. The results showed that treatment of monocytes or THP-1 cells with anti-β2GPI/β2GPI complex could increase TF, MyD88, TRIF as well as TNF-α (tumor necrosis factor alpha) expression. These effects were blocked by addition of TAK-242, a blocker of signaling transduction mediated by the intracellular domain of TLR4. Moreover, TLR4/β2GPI/ANX2 complex could be detected in THP-1 cell lysates. Overall, our results indicate that anti-β2GPI/β2GPI complex induced TF and TNF-α expression involving both TLR4/MyD88 and TLR4/TRIF signaling pathways and TLR4 and its adaptors might be molecular targets for therapy of antiphospholipid syndrome (APS).