Bone reconstruction is essential in orthodontic treatment that caters to the correction of malocclusion by bone reconstruction. Mesenchymal stem cells (MSCs) have been demonstrated a great potency of osteogenesis. The aim of this study was to investigate the effect of hypoxia on the rat bone marrow MSCs (rBMSCs) in vitro during osteogenesis. In this study, we found that temporary exposure of rBMSCs after osteogenic induction for 7 days to hypoxia (2% oxygen) led to a marked decrease in ALPase activity and the expression of osteocalcin and Runt related transcription factor 2/core binding factor a1 (Runx2/Cbfa1). Meanwhile, we found that exposure to hypoxia led to an early and transient increase in the level of phosphorylated ERK1/2 but had no obvious effects on mitogen-activated protein kinase (p38 MAPK) level. Based on these results, we concluded that hypoxia could inhibit osteogenic differentiation of rBMSCs possibly through MEK-ERK 1/2, while p38 MAPK may not participate in this regulation. Further exploration into the mechanisms of hypoxia on osteogenesis would surely provide reliable evidence for clinical practice.