OBJECTIVE: This study aimed to investigate the effects of hypoxia on the characteristics of human dental follicle cells (hDFCs). METHODS: The tissue explant collagenase method was used to isolate hDFCs from young permanent teeth. The immunofluorescence technique was used to detect cell surface markers, and the multi-differentiation potential was detected by multilineage differentiation induction assay. Then, the hypoxic microenvironment was physically mimicked, and the cells were divided into the normoxia group (20%O₂) and the hypoxia group (2%O₂). The effects of hypoxia on cell migration and proliferation were examined by Transwell chamber test and CCK-8 assay, respectively. The gene and protein expression levels of stemness-related markers at both oxygen concentrations were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. After osteogenic induction of both groups, qRT-PCR was performed to evaluate the osteogenesis-related gene, and alizarin red staining was used to assess the formation of mineralized nodules. RESULTS: With the multi-differentiation capacity of osteogenic cells, adipogenic cells, and nerves, hDFCs demonstrate strong stem cell characteristics and possess the criteria of mesenchymal stem cells, which can meet the requirements of seed cells in dental tissue engineering. Hypoxia was conducive to the maintenance of hDFC stemness. Hypoxia promoted the migration and proliferation of hDFCs. The hDFCs were induced to osteogenic differentiation under hypoxic conditions, thereby enhancing osteogenesis. CONCLUSIONS: Hypoxic microenvironment plays an important role in maintaining the stemness and promoting the proliferation, migration, and differentiation of hDFCs. Thus, this microenvironment could also serve several important functions in future clinical applications.