Immunotherapy can effectively suppress tumor, yet complete tumor eradication occurs infrequently. The metastatic potential of remnant tumor cells after immunotherapy and the underlying mechanisms have not been fully elucidated. Here, we report that the termination of immunotherapy strikingly increases the metastatic potential of remnant melanoma. This is mainly due to the withdrawal of IFN-gamma after immunotherapy. The relief of IFN-gamma stress led to the increase of alphavbeta3 integrin expression in B16 cells, which increased the adhesion of B16 cells to fibrinogen, fibronectin and laminin. Through alphavbeta3 signaling, the activation of FAK, upregulation of cdc2, production of active MMP-2 and MMP-9 and actin polymerization were intensified in B16 cells stimulated with ECM molecules 24 h after the withdrawal of IFN-gamma. The i.v. injection of such tumor cells into mice resulted in more metastatic tumor nodes in lung and shortened the survival of mice. The pitfall of immunotherapy termination can be remedied by the administration of recombinant CBD-HepII polypeptide of fibronectin, which effectively inhibits alphavbeta3 signaling. These findings suggest that the risk of tumor metastasis can be increased after the termination of immunotherapy, due to the withdrawal of IFN-gamma and that targeting alphavbeta3 signaling pathway can improve the therapeutic effect of immunotherapeutic approaches by reducing such metastatic risk.