Fractalkine (FKN) has been implicated in modulating angiogenesis and vascular inflammation, but the underlying mechanism has not been elucidated. We here investigated the molecular mechanism by which FKN regulates angiogenesis. We found that recombinant FKN increased in vitro proliferation, migration, and tube formation of human umbilical endothelial cells, as well as stimulated in vivo angiogenesis. FKN-induced angiogenesis was accompanied by phosphorylation of ERK, Akt, and endothelial nitric oxide synthase (eNOS) as well as an increase in NO production. These biochemical events and angiogenesis were completely inhibited by the G protein-coupled receptor inhibitor pertussis toxin. Inhibitors of Raf-1, MEK, PI3K, and eNOS or transfection with dominant negative forms of ERK and Akt significantly suppressed the angiogenic activity of FKN. However, inhibitors of Raf-1 and MEK or a dominant negative ERK mutant blocked FKN-induced ERK phosphorylation, but not Akt and eNOS phosphorylation. The PI3K inhibitor and a dominant negative mutant of Akt suppressed Akt and eNOS phosphorylation and NO production. Our results demonstrated that FKN stimulated angiogenesis by activating both the Raf-1/MEK/ERK and PI3K/Akt/eNOS/NO signal pathways via the G protein-coupled receptor CX3CR1, indicating that two pathways are required for full angiogenic activity of FKN. This study suggests that FKN may play an important role in the pathophysiological process of inflammatory angiogenesis.