Edible grey oyster mushroom, Pleurotus sajor-caju, β (1,3), (1,6) glucan possesses a wide range of biological activities, including anti-inflammation, anti- microorganisms, and antioxidant. However, its biological activity is limited by their low water solubility resulting from the high molecular weight. Our previous study demonstrated that enzymatic hydrolysis of grey oyster mushroom β-glucan using Hevea β-1,3-glucanase isozymes obtains a lower molecular weight and higher water solubility, Pleurotus sajor-caju glucanoligosaccharide (Ps-GOS). Additionally, Ps-GOS potentially reduces osteoporosis by enhancing osteoblast-bone formation, whereas its effect on osteoclast-bone resorption remains unknown. Therefore, our study investigated the modulatory activities and underlying mechanism of Ps-GOS on RANKL-induced osteoclastogenesis in pre-osteoclastic RAW 264.7 cells. Cell cytotoxicity of Ps-GOS on RAW 264.7 cells was determined by the MTT assay and its effect on osteoclast differentiation was determined by TRAP staining. Additionally, its effect on osteoclast bone-resorptive ability was detected by the pit formation assay. The osteoclastogenic-related factors were assessed by qRT-PCR, western blot and immunofluorescence. The results revealed that Ps-GOS was non-toxic and significantly suppressed the formation of mature osteoclast multinucleated cells and their resorption activity by reducing the number of TRAP-positive cells and pit formation areas in a dose-dependent manner. Additionally, Ps-GOS attenuated the NFB-P65 expression and its subsequent master osteoclast modulators including NFATc1 and cFOS via the NF-B pathway. Furthermore, Ps-GOS markedly inhibited RANK expression, which serves as an initial transmitter of many osteoclastogenesis-related cascades, and inhibited proteolytic enzymes, including TRAP, MMP-9, and CTK. These findings indicate that Ps-GOS could potentially be beneficial as an effective natural agent for bone metabolic disease.