In order to investigate the effects of hyperbaric oxygen (HBO) on the proliferation and differentiation of human osteoblasts isolated from alveolar bone under various pressure and exposure time, osteoblasts from human alveolar bone were seeded in 24 well plates at a cell density of 2 500 cells per well. There are four treatment groups which were 2.4ATA for 90 min, 2.4ATA for 30 min, 1.5ATA for 90 min and 1.5ATA for 30 min. Osteoblasts culture were treated one time everyday for up to 10 days in a temperature and humidity controlled custom-made seven-litre hyperbaric unit. Control samples were incubated in a standard humidified incubator at 37℃ containing 5% CO₂ and 95% atmospheric air. Proliferation of osteoblasts were evaluated by WST-1 assay before and 16 h after HBO on day 1,2,3,4,6,8,10. The cytotoxic effect of HBO on osteoblasts was assessed by a toxicology assay kit. For differentiation study, osteoblasts were seeded in 96 well plates at a cell density of 10 000 cells per well. After 3 days normal culture, medium was changed to osteogenic medium. Subsequently, cultures were exposed daily to HBO of 2.4ATA for 90 min and 1.5ATA for 90 min up to 13 days. Mineralization was evaluated by calcium deposition assay, alkaline phosphatase (ALP) activity and von Kossa staining. To assess the effect of pressure on cell proliferation and differentiation, hyperbaric air treatment was observed in this study. It showed that HBO treatment promotes proliferation of osteoblast in the presence of 10% foetal calf serum (FCS). No significant change in extracellular LDH activity before and after HBO treatment. The study of differentiation demonstrated that HBO enhanced differentiation associated with increased bone nodule formation, calcium deposition and alkaline phosphatase activity. These result suggests that HBO treatment significantly stimulated osteogentic differentiation, which implies a potential application of HBO in bone tissue engineering.