The purpose of this study was to investigate the effects of water extracts from edible mushroom, Pleurotus citrinopileatus fruiting body on RANKL-induced osteoclast differentiation. The osteoclast differentiation inhibitor from water extracts of P. citrinopileatus was purified by ultrafiltration, size exclusion column chromatography, ethanol precipitation, and treatment with various enzymes. The water extracts from P. citrinopileatus showed significant osteoclast differentiation inhibitory activity, and its 50 kDa above fraction from ultrafiltration showed the highest osteoclast differentiation inhibitory activity. Fractions 2 and 3 from size exclusion column chromatography clearly inhibited RANKL-induced osteoclast differentiation. When these fractions were treated by ethanol precipitation, the precipitates showed high osteoclast differentiation inhibitory activity. Finally, we obtained the precipitates as the purified osteoclast differentiation inhibitor, and it was identified as β-glucan. However, further studies are required to be used as candidate of anti-osteoporotic agent.