Toll-like receptor 3 (TLR3) is a member of the Toll-like receptors which recognize pathogen-associated molecular patterns leading to the activation of the innate immune response. Recent reports have strongly indicated that they play important roles in cancer cells. Since TLR3 has been recently suggested as a possible therapeutic target in certain types of cancers, in the present study, TLR3 expression and its function were explored in hepatocellular carcinoma (HCC) and human umbilical vein endothelial cells (HUVECs). The expression of TLR3 in various HCC cell lines and HUVECs was detected using quantitative real-time PCR (qRT-PCR) and immunocytochemistry. TLR3 activity was determined by Luciferase reporter assays. The effects of TLR3 double-stranded RNA (dsRNA) agonists on angiogenesis were tested by aortic ring assay and HUVEC tube formation experiments. After dsRNA treatment, cell apoptosis was assessed by Annexin V and PI staining through FACS, and the migration ability was measured by a migration assay. The results showed that TLR3 was expressed in HCC cell lines and HUVECs at the mRNA and protein level. Luciferase reporter assays demonstrated that TLR3 was activated by the dsRNA analog BM-06 or poly(I:C). Rat aortic ring outgrowth and endothelial cell tube formation were suppressed after treatment with dsRNA. In addition, dsRNA triggered apoptosis in MHCC97H, SMMC-7721 and HUVEC cell lines and inhibited cell migration. In conclusion, TLR3 agonists not only affect tumor microenvironment by suppressing angiogenesis but also directly induce tumor cell apoptosis and inhibit tumor cell migration. TLR3 may be a new target for HCC therapy.