Objective: The present study investigated the detailed mechanism by which fractalkine (Fkn), a CX3C chemokine, induces angiogenesis and its functional implication on alleviating ischemic condition in vivo. Methods and Results: Fkn induced new vessel formation on the excised rat aorta and chick���s chorioallantoic membrane (CAM) through CX3CR1 activation. Immunoblotting analysis and EMSA showed that Fkn upregulated hypoxia-inducible factor-1 alpha (HIF-1慣) by cultured human aortic endothelial cells (HAECs), which in turn induced mRNA and protein productions of VEGF-A, a dominant isoform of vascular endothelial growth factor (VEGF), through p42/44 MAPK pathway. In vivo Fkn-induced angiogenesis on CAM was completely abrogated by functional inhibition of VEGF receptor 2 (KDR) and Rho GTPase. Overt angiogenesis was developed in the Fkn-mixed Matrigel plug implanted to C57/BL6 mice with CX3CR1(-/-) bone marrow-derived cells, suggesting CX3CR1 activation in ECs is sufficient for Fkn-induced angiogenesis in vivo. The condition of rat hindlimb ischemia, which rapidly stimulated mRNA expressions of both Fkn and VEGF-A, was significantly improved by injection of whole-length Fkn protein. Conclusions: Fkn-induced activation of CX3CR1 by ECs induces in vivo angiogenesis through two sequential steps; the induction of HIF-1慣 and VEGF-A gene expression after CX3CR1 activation, and the subsequent VEGF-A/KDR-induced angiogenesis. The potent induction of angiogenesis by Fkn can be used as a therapeutic strategy for alleviating peripheral ischemia.