Severe malocclusion can contribute to several serious dental and physical conditions, such as digestive difficulties, periodontal disease, and severe tooth decay. Orthodontic treatment is mainly used to treat malocclusion. Forces in orthodontic tooth results in bone resorption on the pressure side and bone deposition on the tension side. Osteoblasts have been considered as the key component in bone regeneration on the tension side. However, the underlying mechanisms remain unclear. In this study, we focus on how mechanical stretch regulates the osteogenesis during orthodontic treatment. Human jaw bone marrow mesenchymal stem cells (hJBMMSCs) were isolated from healthy adult donors and cultured in regular medium (control) or osteogenic medium (OS). Under OS culture, hJBMMSCs presented osteogenic differentiation potentials, as evidenced by increased mineralization, enhanced calcium deposition, and upregulated expression of osteogenesis markers (ALP, osterix, and Runx). What's more, the OS-induced osteogenesis of hJBMMSCs is associated with the dephosphorylation of IKK, activation of IKBα, and phosphorylation/nucleic accumulation of P65, which all indicated the inhibition of NF-κB activity. Overexpressing P65 in hJBMMSCs, which could constantly activate NF-κB, prevented the osteogenic differentiation in the OS. After that, we applied the Flexcell tension system, which could cause mechanical stretch on cultured hJBMMSCs to mimic the tension forces during tooth movement. Mechanical stretch resulted in 3.5-fold increase of ALP activity and 2.4-fold increase of calcium deposition after 7 days and 21 days treatment, respectively. The expression levels of ALP, Run×2, and Osterix were also significantly upregulated. In the meantime, applying mechanical stretch on OS-cultured hJBMMSCs also dramatically promoted the OS-induced osteogenesis. Both OS and mechanical stretch downregulated NF-κB activity. By overexpressing P65 in hJBMMSCs, neither OS nor mechanical stretch could induce their osteogenesis. These results indicated that, like OS induction, mechanical stretch-facilitated osteogenesis of hJBMMSCs by inhibiting NF-κB in the noninflammatory environments.