Macrophage infiltration is observed during the course of corneal neovascularization. We examined corneal neovascularization process after alkali injury in mice deficient in fractalkine receptor/CX3CR1, which is expressed by macrophages. The mRNA expression of CX3CR1 and its ligand CX3CL1/fractalkine was augmented together in the corneas after alkali injury. Compared with WT mice, CX3CR1‐deficient (KO) mice exhibited enhanced corneal neovascularization 2 weeks after injury, as evidenced by enlarged CD31‐positive areas. Concomitantly, the infiltration of F4/80‐positive macrophages but not Gr‐1‐positive neutrophils was significantly attenuated in KO mice comparing with WT mice. The intraocular mRNA expression of angiogenic factor, vascular endothelial factor, was enhanced to similar extents in WT and KO after the injury. In contrast, the mRNA expression of anti‐angiogenic factors, a disintegrin and metalloprotease with thrombospondin (ADAMTS)‐1 and thrombospondin (TSP)‐1 was enhanced in wild‐type mice but the enhancement was attenuated in KO mice. A double immunofluorescence analysis demonstrated that F4/80‐positive cells also expressed CX3CR1 and ADAMTS‐1, TSP‐1 and ADAMTS‐1 were detected in CX3CR1‐positive cells. CX3CL1 enhanced the ADAMTS‐1 and TSP‐1 expression by peritoneal macrophages. Moreover, topical application of CX3CL1 inhibited corneal neovascularization at 2 weeks, together with enhanced intraocular ADAMTS‐1 and TSP‐1 expression. Thus, these observations indicate that intraocular infiltrating CX3CR1‐positive macrophages can dampen alkali‐induced corneal neovascularization by producing anti‐angiogenic factors, such as ADAMTS‐1 and TSPs.