The innate immune system builds up host defense against a huge diversity of pathogens, such as viruses, bacteria and fungi. Toll-like receptors (TLRs) are one of the pattern recognition receptors that trigger the initiation of various defense mechanisms. The expression of TLRs represents an important link between innate and adaptive immune responses. The expression of TLRs in several cancer cell types has been reported; however, the exact roles they play in cancers are still unclear. In this study, we investigated the expression and signaling of TLRs in human head and neck squamous cell carcinomas (HNSCCs). The expression of TLRs in 8 HNSCC cell lines was examined by Western blot analysis and flow cytometry. The expression of TLR mRNA was also examined by quantitative RT-PCR. Cells were incubated with poly I:C, a TLR3 ligand, and cell viability was tested by MTT assay. In addition, apoptosis was analyzed by flow cytometry with Annexin V and propidium iodide staining. The expression of survivin, a member of the inhibitors of the apoptosis protein family, was also investigated. TLR2 and TLR3 were widely expressed in human HNSCCs. Interestingly, the stimulation of TLR3 by poly I:C induced apoptosis in cancer cells in a dose-dependent manner. The expression of survivin was down-regulated during apoptosis, suggesting that TLR signaling affects survivin-mediated signal transduction in apoptosis. In the present study, we demonstrated the proapoptotic activity of TLR3 expressed by HNSCCs. These results suggest that TLR3 could be a new target for therapy in HNSCCs.