Toll-like receptors (TLRs) are an essential component of the innate immune response to microbial pathogens. TLR3 is localized in intracellular compartments such as endosomes and signals in response to virus-derived double-stranded RNA (dsRNA). TLR3 localization within endosomes is required for ligand recognition, suggesting that acidic pH is the driving force for TLR3 ligand binding. To clarify the pH-dependent binding mechanism of TLR3 at the structural level, we focused on 3 highly conserved histidine residues clustered at the N-terminal region of the TLR3 ectodomain (ECD): H39, H60 and H108. Mutagenesis of these residues showed that H39, H60, and H108 were essential for ligand-dependent TLR3 activation in a cell-based assay. Furthermore, dsRNA binding to the recombinant TLR3 ECD depended strongly on pH and dsRNA length, and was reduced by mutations of H39, H60, and H108, demonstrating that TLR3 signaling is initiated from the endosome through a pH-dependent binding mechanism, and that a second dsRNA binding site exists in the N-terminal region of the TLR3 ECD. We propose a novel model for the formation of TLR3 ECD dimers complexed with dsRNA that incorporates this second binding site.