Monocyte migration into diseased tissues contributes to the pathogenesis of diseases. Intermediate-conductance Ca2+-activated K+ (KCa3.1) channels play an important role in cell migration. However, the role of KCa3.1 channels in mediating monocyte migration induced by palmitic acid (PA) is still unclear. Using cultured THP-1 cells and peripheral blood mononuclear cells from healthy subjects, we investigated the role and signaling mechanisms of KCa3.1 channels in mediating the migration induced by PA. Using methods of Western blotting analysis, RNA interference, cell migration assay and ELISA, we found that PA-treated monocytes exhibited increment of the protein levels of KCa3.1 channel and monocyte chemoattractant protein-1 (MCP-1), and the effects were reversed by co-incubation of PA with anti-TLR2/4 antibodies or by specific inhibitors of p38-MAPK, or NF-κB. In addition, PA increased monocyte migration, which was abolished by a specific KCa3.1 channel blocker, TRAM-34, or KCa3.1 small interfering RNA (siRNA). The expression and secretion of MCP-1 induced by PA was also similarly prevented by TRAM-34 and KCa3.1 siRNA. These results demonstrate for the first time that PA upregulates KCa3.1 channels through TLR2/4, p38-MAPK and NF-κB pathway to promote the expression of MCP-1, and then induce the trans-endothelial migration of monocytes.