Lipopolysaccharide (LPS) can induce bone loss by stimulating osteoclast formation. Colony-stimulating factor 1 receptor (CSF 1R) inhibitors have great potential for the treatment of rheumatoid arthritis and tumor-related bone erosion. However, its role in LPS-induced bone loss is still not clarified. In this study, we observed the effects of CSF 1R inhibitor, PLX3397, on LPS-induced bone damage in an animal model. The models were established by LPS administration in male Sprague-Dawley rats. PLX3397 (30 mg/kg body weight) was given by oral gavage. MicroCT analysis, biomechanical properties, biomarker assay, histological examination, and mRNA expression of osteoclast differentiation-related genes (Traf6, Fra1, c-fos and NFATc1) were performed on the 8th week. LPS induced bone loss was shown as the decrease in bone volume fraction and trabecular number and increase in trabecular separation (p < 0.05). LPS exposure also markedly decreased the bone biomechanical properties. PLX3397 significantly abolished the LPS-induced bone microstructure damage (p < 0.05) and loss of biomechanical properties. PLX3397 also inhibited the increases of serum tartrate-resistant acid phosphatase 5b level enhanced by LPS (p < 0.05). PLX3397 attenuated the high expression of Traf6, Fra1, c-fos and NFATc1 stimulated by LPS. Our data demonstrated that PLX3397, a type of CSF 1R inhibitor, can suppress LPS-induced bone loss via the inhibition osteoclast formation.