Pattern recognition receptors (PRRs) play a crucial role in the innate immune system and contribute to host defense against microbial infection. PRR-mediated antimicrobial signals provide robust type-I IFN/cytokine production and trigger inflammation, thereby affecting tumor progression and autoimmune diseases. Accumulating evidence demonstrates that among the PRRs, only the signaling pathway of endosomal toll-like receptor 3 (TLR3) induces no systemic inflammation and mediates cross-priming of antigen-specific CD8+ T cells by dendritic cells. Treatment with a newly developed TLR3-specific ligand, ARNAX, along with tumor-associated antigens (TAAs), induces tumor-specific cytotoxic T lymphocytes, modulates the tumor microenvironment to establish Th1-type antitumor immunity, and leads to tumor regression without inflammation in mouse tumor models. Combination therapy using ARNAX/TAA and PD-1/PD-L1 blockade potently enhances antitumor response and overcomes anti-PD-1/PD-L1 resistance. In this review, we will discuss the TLR3-mediated signaling in antitumor immunity and its application to cancer immunotherapy.