Objective Bacterial DNA is a pathogen-derived molecule which can regulate the innate immune system by stimulating NF-κB activation. The activity of bacterial DNA relies on its content of unmethylated CpG dinucleotides in particular base contexts("CpG motif"). In light of the pivotal role played by NF-κB in osteoclast differentiation, the ability of CpG oligodeoxynucleotides (CpG ODN) coming from bacterial DNA to modulate osteoclastogenesis was studied. Methods Bone marrow mononuclear cells (BMM) were purified from Balb/c mice, cultured in α-MEM media containing 10% FCS in the presence of mouse M-CSF, with either RANKL or ODNs for 5 days. Osteoclast formation was evaluated on day 5 according to TRAP and May-Grunwald-Giemsa staining. Results CpG ODN alone could induce osteoclast formation in the low degree in BMM culture. The relationship between CpG ODN and RANKL was that CpG ODN could inhibit RANKL-induced osteoclastogenesis when present from the beginning of BMM culture, but strongly increased RANKL-induced osteoclastogenesis in RANKL-pretreated BMMs. Conclusion The mechanism of CpG ODN regulating osteoclast differentiation was bidirectional, which might be a potential therapy for treating metabolic bone disease.