Primarily, Toll-like receptor 9 (TLR9) is a specific receptor for microbial DNA in human immune cells. TLR9 has been found to be a promising target in tumor immunotherapy but the direct effect of its activation on tumor cells remains unknown. In this study, we examined the effect of TLR9 ligation on estrogen receptor alpha (ERalpha)-mediated transactivation of breast cancer. Luciferase report gene assays, RNA interference of TLR9 and Chromatin immunoprecipitation were performed to measure the effect of TLR9 ligation on ERalpha-mediated transactivity of T47D and MCF-7 cells. Bromodeoxyuridine incorporation assay was used to examine the effect of TLR9 ligation on estrogen (E2)-induced proliferation of breast cancer cells. We also investigated the mechanism for the effect of TLR9 ligation on ERalpha-mediated transactivity. We found that ERalpha-mediated transcription via estrogen response element of human breast cancer cells line T47D was significantly suppressed when treated with 17beta-estradiol in combination with TLR9 agonist CpG oligonucleotides and this effect of CpG was dependent on TLR9. Furthermore, nuclear factor kappaB (NF-kappaB) inhibitor BAY 11-7082 could abolish the inhibitory effect of CpG oligonucleotides on ERalpha-mediated transactivation. We also confirmed the effect of CpG oligonucleotides on ERalpha-mediated transactivation in the breast cancer cell line MCF-7 forced to stably overexpress TLR9. Finally, we observed that CpG oligonucleotides were also able to inhibit estrogen-induced proliferation of breast cancer cells as a consequence of decreased ERalpha-mediated transactivation. Taken together, our data suggest that TLR9 signal pathway, by activating NF-kappaB, negatively regulates ERalpha-mediated transactivation of breast cancer. Thus, TLR9 agonist inhibits the proliferation of breast cancer cells in response to estrogen.