Abstract Many cancer cells express Toll-like receptors (TLR) that offer possible therapeutic targets. Polyadenylic-polyuridylic acid [poly(A:U)] is an agonist of the Toll-like receptor TLR3 that displays anticancer properties. In this study, we illustrate how the immunostimulatory and immunosuppressive effects of this agent can be uncoupled to therapeutic advantage. We took advantage of two TLR3-expressing tumor models that produced large amounts of CCL5 (a CCR5 ligand) and CXCL10 (a CXCR3 ligand) in response to type I IFN and poly(A:U), both in vitro and in vivo. Conventional chemotherapy or in vivo injection of poly(A:U), alone or in combination, failed to reduce tumor growth unless an immunochemotherapeutic regimen of vaccination against tumor antigens was included. CCL5 blockade improved the efficacy of immunochemotherapy, whereas CXCR3 blockade abolished its beneficial effects. These findings show how poly(A:U) can elicit production of a range of chemokines by tumor cells that reinforce immunostimulatory or immunosuppressive effects. Optimizing the anticancer effects of TLR3 agonists may require manipulating these chemokines or their receptors. Cancer Res; 70(2); 490–500