CX3CL1 chemokine (fractalkine) is highly expressed by vascular smooth muscle cells (VSMCs) in atherosclerotic lesions. Its membrane-bound form promotes cell-cell interactions, whereas the soluble form induces chemotaxis of CX3CR1- expressing leukocytes. We show that the cysteine protease cathepsin S, expressed by VSMCs, is able to cleave membrane-anchored CX3CL1, releasing a 55-kDa fragment to the medium, thus regulating the adhesion of VSMCs and the capture of monocytes to the sites of atherogenesis. Moreover, strong co-localization of cathepsin S and CX3CL1 with a recycling endosome marker Rab11a suggests a processing of CX3CL1 in recycling endosomes during its redistribution to the plasma membrane.