Abstract A rational monoclonal antibody (mAb)-based antitumor therapy approach has previously been shown to eradicate various established experimental and carcinogen-induced tumors in a majority of mice. This therapy comprised an agonistic mAb reactive with tumor necrosis factor–related apoptosis-inducing ligand receptor (DR5), expressed by tumor cells, an agonistic anti-CD40 mAb to mature dendritic cells, and an agonistic anti-4-1BB mAb to costimulate CD8+ T cells. Because agonists of CD40 have been toxic in patients, we were interested in substituting anti-CD40 mAb with other dendritic cell–maturing agents, such as glycolipid ligands recognized by invariant natural killer T (iNKT) cells. Here, we show that CD1d-restricted glycolipid ligands for iNKT cells effectively substitute for anti-CD40 mAb and reject established experimental mouse breast and renal tumors when used in combination with anti-DR5 and anti-4-1BB mAbs (termed “NKTMab” therapy). NKTMab therapy–induced tumor rejection was dependent on CD4+ and CD8+ T cells, NKT cells, and the cytokine IFN-γ. NKTMab therapy containing either α-galactosylceramide (α-GC) or α-C-galactosylceramide (α-c-GC) at high concentrations induced similar rates of tumor rejection in mice; however, toxicity was observed at the highest doses of α-GC (>250 ng/injection), limiting the use of this glycolipid. By contrast, even very low doses of α-c-GC (25 ng/injection) retained considerable antitumor activity when used in combination with anti-DR5/anti-4-1BB, and thus, α-c-GC showed a considerably greater therapeutic index. In summary, sequential tumor cell apoptosis and amplification of dendritic cell function by NKT cell agonists represents an exciting and novel approach for cancer treatment. [Cancer Res 2007;67(15):7495–504]