Macrophages in tumor microenvironment have pivotal roles in tumor growth, metastasis, and angiogenesis. We investigated the interacting mechanism of macrophage actions in human papillary thyroid cancer (PTC). Co-cultures of macrophage/PTC significantly increased the cancer cell migration potentials, compared with the PTC culture alone. Treatment of conditioned medium (CM) of macrophage/PTC co-cultures enhanced cell invasions in 3D invasion assay. Cytokine array analysis demonstrated that CM of macrophage/PTC co-cultures contained a high level of CXCL16, while it was not found in CM of PTC culture alone. Treatment with CXCL16 enhanced the cell migration potentials in PTC cells, and blocking CXCL16 signaling using anti-CXCL16 antibody or metalloproteinase inhibitor (TAPI2) attenuated macrophage-mediated enhancement of PTC cell migration potentials. In PTC cells, CXCL16 treatment or co-cultures with macrophages increased Akt phosphorylation, and these macrophage-dependent increases of Akt phosphorylation was inhibited by anti-CXCL16 antibody. Moreover, Akt inhibitor attenuated macrophage-mediated increases of PTC cell migration potential. In macrophages, treatment of macrophage/PTC co-cultured CMs up-regulated CD163, Il10, and CD206, which were attenuated by anti-CXCL16 antibody treatment. Finally, CXCR6 and CXCL16 expressions were evaluated by immunohistochemical staining with a thyroid tissue microarray including 136 PTC. CXCR6 expressions showed positive correlation with the density of CD163(+) macrophages and associated with lymph node metastasis. In conclusion, CXCL16 signaling partly mediated macrophage actions on PTC tumor cell invasion and also changed the macrophage phenotypes into M2-macrophages in PTC tumor microenvironment. These data suggested that CXCL16 signaling, a bidirectional player in macrophage-associated tumor microenvironment, might be a potential therapeutic target of human PTC.