Abstract Cancer vaccine-based immunotherapy is emerging as a novel therapeutic strategy for cancer treatment. However, its antitumor effect remains unsatisfied due to the poor immunogenicity of tumor antigens (Ags). Although polyriboinosinic: polyribocytidylic acid (PIC), a TLR3 agonist, has been reported as a promising adjuvant for cancer vaccines, its immunopotency may be limited by insufficient cellular penetration. In the present study, we incorporated PIC into DOTAP cationic liposome to generate PIC–DOTAP Liposome Complex (PDLC) nanoparticles. The results showed that PDLC was more potent than DOTAP or PIC to enhance vaccine-induced tumor-specific cytotoxic T lymphocyte (CTL) response and IFN-γ production. Moreover, two doses of PDLC vaccines remarkably suppressed tumor growth in mice, which involved the participance of CD8+ T cells and depended on the presence of Ag. The superior antitumor effect of PDLC vaccines could be attributable to enhanced maturation of mouse bone-marrow dendritic cells (BMDCs) and increased production of type I IFNs. More importantly, PDLC strengthened the TLR3 signaling in BMDCs by enhancing the interaction of PIC with TLR3 and augmenting downstream IRF-3 phosphorylation, as well as elevating IRF-3/IRF-7 mRNA transcription. Taking together, the complex of PIC and DOTAP liposomes enhanced PIC uptake and consequential TLR3 signaling in BMDCs, which in turn promoted DC maturation and type I IFN production, thereby augmenting the antitumor effect of cancer vaccines.