Objective This study aimed to explore the effect of the up-regulation of Notch1 on osteoclastogenesis induced to osteoclasts by receptor activator for nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factors (MCSF)in vitro.Methods The bone marrow stem cells (BMSCs) of Rosa-notch1 mice were cultured and induced to osteoclasts by RANKL and MCSF. The BMSCs were transfected with the Ad-Cre-green fluorescent protein (GFP) virus or Ad-GFP virus. Total RNA from cells was extracted, and the gene expression levels of Notch1, Notch2, Notch3, Notch4, Delta1, Delta3, Delta4, Jagged1, Hes1, and tartrate resistant acid phosphatase (TRAP) were detected at the defined stage by reverse transcription-polymerase chain reaction (RT-PCR). Osteoclast formation was analyzed by TRAP assay.Results The number of TRAP-positive multinuclear cells of the experimental group significantly decreased compared with that of the control group. The mRNA expression levels of Notch1, Notch3, Jagged1, Delta3, and Hes1 of the experimental group were significantly higher than those of the control group, whereas the TRAP mRNA expression of the experimental group was significantly lower than that of the control group (PConclusion Up-regulation of Notch1 inhibit osteoclastogenesis of BMSCs induced by RANKL and MCSF in vitro.