The chemokine receptor CCR2 controls the release of Ly6C(high) monocytes from the bone marrow and their recruitment to sites of inflammation. A second chemokine receptor, CX3CR1, is differentially expressed on monocyte subsets. We examined the role of CX3CR1 in monocyte trafficking during the recovery phase after cyclophosphamide (CP)-induced myeloablation and observed that, in the absence of CCR2, Ly6C(high) monocytes accumulated in the bone marrow and peripheral reconstitution was severely impaired compared to wild-type (WT) mice. Contrastingly, in the absence of CX3CR1, Ly6C(high) monocytes accumulated less rapidly in the marrow but recovered faster in the blood and were more recruited into the spleen, suggesting opposite action between CCR2 and CX3CR1 in the myelorestoration. During the recovery phase, marrow medullar monocytes displayed lower CX3CR1 expression and reduced their adherence to coated CX3CL1. Intravital imaging of the bone marrow showed that CP treatment impacts monocyte trafficking between the parenchyma and the vasculature. Medullar monocytes in CX3CR1(-/-) mice, and mice treated with a specific antagonist of CX3CR1, displayed increased mean velocity and displacement and reduced their arrest coefficient compared to WT mice. This study indicates that CX3CR1 reduces the motility of Ly6C(high) monocytes in the bone marrow and thereby controls their release.