OBJECTIVES: Osteoblasts play an important role in the process of osteogenesis and prevention of osteonecrosis. Dexamethasone, a type of glucocorticoids (GCs), induce apoptosis of osteoblasts and lead to the occurrence of non-traumatic osteonecrosis. This study aimed to explore the effects of different doses and duration of Dexamethasone on osteoblast apoptosis of rats in vitro. METHODS: Proliferation and apoptosis of osteoblasts after Dexamethasone treatment were detected using cell counting kit-8 (CCK-8) assay and FITC-Annexin V/PI staining. The expressions of caspase-3 and -9 in osteoblasts after Dexamethasone treatment were analyzed using western blotting and qRT-PCR. Dexamethasone remarkably inhibited proliferation and induced apoptosis of osteoblasts in a dose-and duration-dependent manner. RESULTS: As the intervention time extended, the expression of caspase-3 mRNA and caspase-9 mRNA in different Dexamethasone groups gradually increased in a duration-dependent manner. With the same time of intervention (12h, 24h, 48h), the expression of caspase-3 and -9 mRNA gradually increased in a dose-dependent manner. After treated with 5 * 10-8M, 5 * 10-7M, 5 * 10-6M and 5 * 10-5M Dexamethasone for 24 hours, the expression of cleaved caspase-3 and -9 protein increased in a dose-dependent manner. CONCLUSION: Dexamethasone can induce osteoblast apoptosis in a duration- and dose-dependent manner.