Bone loss in Staphylococcus aureus (S. aureus) osteomyelitis poses a serious challenge to orthopedic treatment. The present study aimed to elucidate how S. aureus infection in bone might induce bone loss. The C57BL/6 mice were injected with S. aureus (106 CFU/ml, 100 μl) or with the same amount of vehicle (control) via the tail vein. Microcomputed tomography (microCT) analysis showed bone loss progressing from week 1 to week 5 after infection, accompanied by a decreased number of osteocalcin-positive stained osteoblasts and the suppressed mRNA expression of Runx2 and osteocalcin. Transcriptome profiles of GSE30119 were downloaded and analyzed to determine the differences in expression of inflammatory factors between patients with S. aureus infected osteomyelitis and healthy controls, the data showed significantly higher mRNA expression of granulocyte colony-stimulating factor (G-CSF) in the whole blood from patients with S. aureus infection. Enzyme-linked immunosorbent assay (ELISA) analysis confirmed an increased level of G-CSF in the bone marrow and serum from S. aureus infected mice, which might have been due to the increased amount of F4/80+ macrophages. Interestingly, G-CSF neutralizing antibody treatment significantly rescued the bone loss after S. aureus infection, as evidenced by its roles in improving BV/TV and preserving osteocalcin- and osterix-positive stained cells. Importantly, we found that G-CSF level was significantly up-regulated in the serum from osteomyelitis patients infected by S. aureus. Together, S. aureus infection might suppress the function of osteoblastic cells and induce progressive bone loss by up-regulating the level G-CSF, suggesting a therapeutic potential for G-CSF neutralization in combating bone loss in S. aureus osteomyelitis.