Increasing evidence suggests that malignant transformation can result from chronic infection, and Toll-like receptors (TLRs) may play an important role in this process. We have previously reported that the increased expression of TLR-9 is associated with tumor cell proliferation in oral cancer. However, the mechanisms involved have not been elucidated. The aim of this study was to investigate whether CpG-oligodeoxynucleotides (CpG-ODN), a special TLR-9 agonist, is able to exert the proliferation-promoting effect in human oral squamous cell carcinoma (OSCC), and to explore the possible underlying molecular mechanism. Flow cytometry, MTT, and colony formation assay were used to evaluate cell proliferation and cell cycle distribution. The mRNA and protein levels were analyzed by quantitative RT-PCR and Western blot assay. Luciferase reporter gene, EMSA, and ChIP assays were used to detect the activity of activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) in HB cells. Results showed that CpG-ODN could stimulate proliferation of HB cells in a dose- and time-dependent manner with a promoted G1/S cell cycle progression. Increased cyclin D1 expression was detected in the nuclear region after CpG-ODN treatment. Moreover, CpG-ODN promoted nuclear translocation and activation of AP-1, which appeared to be required for TLR-9-mediated cyclin D1 expression and subsequently cell proliferation, but seemed to have little impact on NF-κB activity. Our results indicate that CpG-ODN stimulates tumor cell proliferation through TLR-9-mediated AP-1-activated cyclin D1 expression in OSCC HB cells. Pharmacologic inhibition of the TLR-9/AP-1/cyclin D1 pathway may be a new therapeutic approach for prevention as well as treatment of OSCC.