Certain types of cationic lipids have shown promise in cancer immunotherapy， but their mechanism of action is poorly understood. In this study， we describe the properties of an immunotherapeutic consisting of the pure cationic lipid enantiomer R-1，2-dioleoyl-3-trimethyl-ammonium-propane (R-DOTAP) formulated with modified viral or self-peptide Ags. R-DOTAP formulations with peptide Ags stimulate strong cross-presentation and potent CD8 T cell responses associated with a high frequency of polyfunctional CD8 T cells. In a human papillomavirus tumor model system， a single s.c. injection of tumor-bearing mice with R-DOTAP plus human papillomavirus Ags induces complete regression of large tumors associated with an influx of Ag-specific CD8 T cells and a reduction of the ratio of regulatory/Ag-specific CD8 T cells. R-DOTAP also synergizes with an anti-PD1 checkpoint inhibitor， resulting in a significant inhibition of B16 melanoma tumor growth. We found that R-DOTAP stimulates type I IFN production by dendritic cells in vivo and in vitro. s.c. injection of R-DOTAP results in an IFN-dependent increase in draining lymph node size and a concomitant increase in CD69 expression. Using knockout mice， we show that type I IFN is required for the induction of CD8 T cell activity following administration of R-DOTAP plus Ag. This response requires Myd88 but not TRIF or STING. We also show that R-DOTAP stimulates both TLR7 and 9. Collectively， these studies reveal that R-DOTAP stimulates endosomal TLRs， resulting in a Myd88-dependent production of type I IFN. When administered with Ag， this results in potent Ag-specific CD8 T cell responses and antitumor activity.