OBJECTIVE: Receptor activator of nuclear factor-κB ligand (RANKL) is intimately involved in regulating bone remodeling during osteoclast differentiation and promotion of osteoclast function. Upon binding to its receptor, RANK, RANKL activates various signaling cascades that induce osteoclast differentiation of osteoclast precursor cells into osteoclasts. In the innate immune system, host pattern recognition receptors, such as Toll-like receptors and nucleotide-binding oligomerization domain-like receptors (NLRs), detect pathogen-associated molecular patterns and elicit an immune response. The NLR, nucleotide-binding oligomerization domain 2 (NOD2), is known to bind muramyl dipeptide (MDP) and regulate inflammatory responses via nuclear factor-κB (NF-κB). The objective of this study was to investigate the effect of MDP on RANKL stimulation of osteoclast differentiation to elucidate the mechanism of bone resorption in a bacterial infection-induced inflammation model. METHODS: The extent of osteoclast formation in MDP-stimulated RAW 264.7 cells was assessed using a tartrate-resistant acid phosphatase activity assay. The protein levels of intracellular signaling molecules were assessed by western blotting. RESULTS: In RAW 264.7 cells, MDP stimulation did not affect the expression of RANK. MDP enhanced the expression of osteoclast-specific proteins, such as nuclear factor of activated T cells 1 (NFATc1) and cathepsin K, which are osteoclast differentiation markers, in RANKL-stimulated RAW 267.4 cells. Furthermore, JSH23, an NF-κB inhibitor, suppressed the expression of NFATc1 after co-stimulation with MDP and RANKL. CONCLUSION: MDP promoted osteoclast differentiation in RAW 267.4 cells by upregulating the activators, NF-κB and NFATc1, which are important for osteoclast differentiation, through enhancement of the RANKL signaling pathway.