The effects of hypoxia on the osteogenic potential of mesenchymal stem cells (MSCs) have been previously reported. From these studies, possible factors affecting the association between hypoxia and the osteogenic differentiation of MSCs have been suggested, including hypoxia severity, cell origin and methods of induction. The effect of the duration of hypoxia, however, remains poorly understood. The aim of the present study was to investigate the effect of continuous hypoxia on the induced osteogenesis of MSCs. Rat MSCs were isolated and cultured in vitro. Once the cells had been cultured to passage three, they were switched to 1% oxygen and cultured either with or without osteogenic medium, while cells in the control groups were cultured under normoxia in corresponding conditions. Four osteogenic differentiation biomarkers, runt-related transcription factor 2, osteopontin, osteocalcin and alkaline phosphatase, were analyzed by quantitative polymerase chain reaction and western blotting at defined intervals throughout the culture period. In addition, Alizarin Red staining was used to assess changes in mineralization. The results showed that 1% hypoxia was able to enhance and accelerate the osteogenic ability of the MSCs during the initial phases of differentiation, and the protein expression of certain associated biomarkers was upregulated. However, continuous hypoxia was shown to impair osteogenesis in the latter stages of differentiation. These findings suggest that hypoxia can regulate the osteogenesis of MSCs in a time-dependent manner.