Accumulating data suggested that functional TLR9 was expressed in various tumor cells and TLR9 signaling could enhance the progression of tumor cells. However, the underlying mechanism of TLR9 signaling on the progression of tumors cells remains largely undefined. Our previous study demonstrated that the TLR9 agonist CpG ODNs could significantly enhance the progression of human lung cancer cells in vivo. Here we further evaluated the direct effect of CpG ODNs on the proliferation and cell cycle of human lung cancer cells. Our data showed that TLR9 agonist CpG ODNs could robustly elevate the proliferation and stimulate cell cycle entry of 95D cells in vitro, accompanied by the selectively up-regulated expression of CDK2. Furthermore, we found that down-regulation of CDK2 expression using siRNA against CDK2 could significantly inhibit the enhanced proliferation of 95D cells induced by CpG ODNs. Finally, we investigated that the CpG ODNs could selectively enhance the promoter activity of CDK2. Our findings indicated that TLR9 signaling could selectively up-regulate the expression of CDK2, which was critical for the enhanced proliferation of human lung cancer cells. Our results might provide novel insight into the understanding of functional expression of TLR9 on the progression of tumor cells.