Prostaglandin-E2 receptors (sub type EP2) are known to be activated during various autoimmune inflammatory disorders including rheumatoid arthritis (RA) and may play an essential role in counteracting the extent of bone damage. Recently, we have reported that EP2 antagonists inhibited pro-inflammatory cytokine responses at the transcriptional level using murine monocytic cell line and observed that EP2 antagonists attenuate the upregulation of several inflammatory mediators in vitro. It has been shown that EP2 gene deletion in mouse cultures impairs osteoclast formation. Herein we investigated the anti-osteoclastogenic activity of recently discovered EP2 antagonists using an in vitro osteoclastogenesis model with mouse monocytic cell line (RAW264.7 cells). We observed significantly increased size and number of osteoclasts by both PGE2 and butaprost (highly selective EP2 agonist) compared to receptor activator of nuclear factor kappa-B ligand (RANKL) alone treated cells. We did not observe significant difference in number of osteoclasts between PGE2 and butaprost. In addition, 10µM of various EP2 specific antagonists inhibited RANKL-induced osteoclast formation; TG8-4 and TG4-155 are more effective than TG6-129. Western blot analysis revealed that EP2 antagonists decreased the expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1), which are the master regulators during osteoclastogenesis. These data suggests the direct effect of EP2 antagonists on bone cells in preventing the severe bone damage. Taken together, these studies indicate that EP2 receptors play a major role during osteoclast formation, thus supporting our study as a therapeutic target to blunt the excessive bone loss during arthritis.