Chemokines are mainly involved in the recruitment of leukocytes into tissues, a key feature of inflammation. Through its unique receptor CX3CR1, the chemokine CX3CL1 participates in diverse inflammatory processes including arterial atherosclerosis and cerebral or renal inflammation. Using a phage display strategy, we engineered a hCX3CL1 analog (named F1) with a modified N terminus. F1 bound specifically to cells expressing hCX3CR1 and had a K(d) value close to that of native CX3CL1. F1 was not a signaling molecule and did not induce chemotaxis, calcium flux, or CX3CR1 internalization. However, it potently inhibited the CX3CL1-induced calcium flux and chemotaxis in CX3CR1-expressing primary cells of human and murine origin with an IC(50) of 5-50 nM. It also efficiently inhibited the cell adhesion mediated by the CX3CL1-CX3CR1 axis. Finally, in a noninfectious murine model of peritonitis, F1 strongly inhibited macrophage accumulation. These data reveal a prototype molecule that is the first bona fide antagonist of hCX3CR1. This molecule could be used as a lead compound for the development of a novel class of anti-inflammatory substances that act by inhibiting CX3CR1.