β-defensins play a dual role during immune response. Their direct antimicrobial properties contribute to the local innate immune response by combating microbial invasions. Furthermore, previous studies revealed the capacity of certain β-defensin family members to chemoattract immature dendritic cells and CD45RO+ CD4+ T cells through chemokine receptor CCR6. However, because β-defensins also chemoattract macrophages and monocytes, which do not express CCR6, efforts have been made to identify other receptors for these polypeptides. In this study, we demonstrate the capacity of human β-defensin (hBD) 2 and 3 and their mouse orthologs, β-defensin 4 and 14, to interact with CCR2, a chemokine receptor expressed on monocytes, macrophages, and neutrophils. These β-defensins, fused to the Fc region of human IgG 1, showed binding to CCR2-transfected HEK293 cells, as revealed by flow cytometry. The β-defensin fusion proteins also induced CCR2-specific chemotaxis of transfected HEK293 cells, human peripheral blood monocytes, and mouse peritoneal exudate cells in a dose-dependent manner. Preincubation of human monocytes with CCL2/MCP-1, the chemokine ligand for CCR2, abolished migration induced by β-defensins. Conversely, preincubation with hBD2: Ig or hBD3: Ig inhibited MCP-1 induced migration. Peritoneal exudate cells from CCR2-deficient mice failed to migrate toward these fusion proteins. In conclusion, the β-defensins used in this study contribute to the innate and adaptive immune response in their role as chemoattractants. Our data indicate that hBD2 and hBD3, together with their mouse orthologs (β-defensin 4 and 14), are chemotactic for a broad spectrum of leukocytes in a CCR6-and CCR2-dependent manner.