To investigate the effect of Staphylococcal peptidoglycan (PGN-sa) on raw264.7 cells differentiating into osteoclasts. There were 5 groups in the experiment: 100 ng/mL PGN-sa group, 200 ng/mL PGN-sa group, 400 ng/mL PGN-sa group, positive control group [100 ng/mL receptor activator of nuclear factor κB ligand (RANKL)], and blank control group (PBS). Raw264.7 cells were cultured with different concentrations of PGN-sa, RANKL, or PBS for 5 days, and then tartrate resistant acid phosphatase (TRAP) staining was used to detect the formation of osteoclast-like cells; Image-Pro Plus 6.0 software was used to detect the bone resorption areas of osteoclast-like cells; and MTT assay was used to observe the proliferation activity of raw264.7 cells. TRAP staining showed that PGN-sa and RANKL can induce raw264.7 cells to differentiate into osteoclast-like cells; different concentrations of PGNsa groups had more osteoclast-like cells formation than blank control group (P<0.05), and the number of osteoclast-like cells significantly increased with the increase of PGN-sa concentrations (P<0.05). Bone resorption cavity experiment showed that bone resorption cavities were obvious in different concentrations of PGN-sa groups and in positive control group, and the area of bone absorption cavities was increased with the increasing PGN-sa concentrations, showing significant difference between groups (P<0.05). MTT assay showed that no significant difference was found in the absorbance (A) value between different concentrations of PGN-sa groups and blank control group, and between different concentrations of PGN-sa groups (P>0.05). PGN-sa can promote raw264.7 cells to differentiate into osteoclasts with bone resorption activity.