Toll-like receptor 9 (TLR9) is a pattern-recognition receptor that is involved in immune signaling and plays a crucial role in cell survival through recognition of various bacterial and viral components including unmethylated CpG-DNA. TLR9 expression and function in cancer cells are not well understood. We investigated the expression of TLR9, and the function of TLR9 signaling, in hepatocellular carcinoma (HCC) cells following stimulation with CpG-oligodeoxynucleotides (ODNs). Positive immunohistochemical staining for TLR9 was observed in 85.7% of HCC tissues. Western blot analysis revealed that TLR9 was expressed both on the cell membrane and in the cytoplasm of HCC cell lines. Full-length TLR9 was predominantly expressed on the membrane rather than in the cytoplasm, whereas multiple cleaved forms of TLR9 were predominantly expressed in the cytoplasm rather than on the membrane. Cell surface stimulation of TLR9 promoted cell proliferation, and, furthermore, the TLR9 agonists, CpG-ODNs, reduced the cytotoxicity of the anti-cancer drug adriamycin (ADM) via up-regulation of apoptosis inhibitors such as survivin, Bcl-xL, XIAP and cFLIP, in HCC cell lines. Although cell surface stimulation of TLR9 did not activate either the NF-kappaB signaling pathway or the type-I IFN secretion pathway, gene chip microarray analysis indicated that TLR9 agonists closely regulated multiple oncology-related genes and transcription factors involved in tumorigenesis and cancer progression. In conclusion, our results indicate that functional cell surface expression of TLR9 in human HCC may play an important role in tumorigenesis and cancer progression.