Thus far, immunotherapies based on one or a few immunostimulatory molecules have shown limited antitumor efficacy. This highlights the need to use multiple immunostimulatory molecules, to target different immune cells, including immunosuppressive cells, simultaneously. Consequently, in this study, we delivered intratumorally via protein transfer four molecules, including the chemotactic molecules secondary lymphoid tissue chemokine and Fas ligand and the costimulatory molecules 4-1BBL and TNF-related activation-induced cytokine. Secondary lymphoid tissue chemokine and Fas ligand together can attract an array of immune cells and induce apoptosis in CD4(+)CD25(+) regulatory T cells (Treg), whereas 4-1BBL and TRANCE together can stimulate T cells and dendritic cells (DCs). We show that the transfer of all four molecules increases tumor-infiltrating neutrophils, DCs, and CD4(+) and CD8(+) T cells and decreases intratumoral Treg. We show that the treatment favors the generation of a Th1 cytokine milieu at the tumor site, which is attributed not only to an increase in IL-12-producting DCs and IFN-gamma-producing CD8(+) T cells, but also to a decrease in IL-10-producing Treg. Importantly, in the L5178Y lymphoma model, we show that compared with transfer of the chemotactic molecules alone or the costimulatory molecules alone, transfer of all four molecules demonstrates stronger antitumor responses against established tumors. Furthermore, we show that the antitumor responses elicited by transfer of all four molecules are mediated by long-term, systemic antitumor immunity. Hence, this study demonstrates for the first time that combinatorial use of chemotactic and costimulatory molecules provides a useful strategy for enhancing antitumor responses.