The objective of this study was to explore the mechanism underlying osteoblast suppression in the process of hematopoietic stem cells mobilization induced by granulocyte colony-stimulating factor (G-CSF). The apoptosis of human and mouse osteoblasts was examined by detecting caspase 3. The levels of serum DKK1 and osteocalcin in the supernatant of co-culture of mouse osteoblasts and mouse bone marrow nucleated cells were measured. The number of mouse osteoblasts co-cultured with mouse bone marrow nucleated cells wasmeasured and the osteocalcin mRNA levelwas also measured. The G-CSF-induced decrease in osteoblast function was partly due to the apoptosis of osteoblasts. There was no significant difference in the level of serum DKK1 in healthy donors before and 5 days after mobilization. The osteocalcin gene and protein expression was significantly different in co-cultured osteoblasts with bone marrow nucleated cells treated with and without G-CSF. Osteoblasts undergo apoptosis during mobilization and G-CSF affects osteoblasts through bone marrow nucleated cells.