AbstractInnate immunity is critically important for tumor surveillance and regulating tumor metastasis. Fractalkine (FKN, CX3CL1), operating through the receptor CX3CR1, is an effective chemoattractant and adhesion receptor for NK cells and monocytes, important constituents of the innate immune response. Previous studies have shown that over‐expression of CX3CL1 by tumor cells enhances antitumor responses. However, since most tumors do not express CX3CL1, it remains unclear if CX3CL1/CX3CR1 has a role in tumor immunity in the absence of ligand over‐expression. To determine the role of CX3CL1 and CX3CR1 in regulating antitumor immune responses, we tested the response of wildtype and CX3CR1‐deficient animals to unmanipulated B16 melanoma that does not express CX3CL1. We studied the distribution and trafficking of mononuclear cells (MNC) under homeostatic conditions and in the presence of B16 metastatic melanoma, cytotoxic activity, and cytokine production in wild‐type and CX3CR1‐deficient animals. We found that B16‐treated CX3CR1−/− mice had increased lung tumor burden and cachexia. There was a selective reduction of monocytes and NK cells in the lungs of CX3CR1‐deficient animals under homeostatic conditions and in response to B16. CX3CR1‐deficient NK cells effectively killed B16 cells in cytotoxicity assays. However, CX3CR1‐deficient NK cells exhibited a tumorigenic cytokine production profile with defective IFN‐γ expression and enhanced IL‐6 production in response to TLR3 activation with polyIC. Our studies indicate that CX3CR1 is an important contributor to innate immunity at multiple levels. Its role in tumor immunity is not limited by expression of CX3CL1 by tumor cells. © 2007 Wiley‐Liss, Inc.