Objective To investigate the effects of vitamin D₃ (VitD₃) on osteoblasts in rats under hypoxia. Methods Newborn rat jaw osteoblasts were extracted and treated with different concentrations of cobalt chloride (0 nmol/mL, 100 nmol/mL, 200 nmol/mL, 300 nmol/mL). The proliferation of osteoblasts at 1, 3 and 5 days was detected by CCK-8 method. Flow cytometry was used to detect the apoptosis rate of osteoblasts on day 3. Osteoblasts were randomly divided into blank control group (0 nmol/mL CoCl₂), CoCl₂ group (300 nmol/mL CoCl₂) and VitD₃ group (300 nmol/mL CoCl₂ + 1 nmol/L VitD₃). VitD₃ group 2(300 nmol/mL CoCl₂ + 5 nmol/L VitD₃). The proliferative changes of osteoblasts at day 1, 3 and 5 under hypoxia were detected by CCK-8 method, and the expression of genes related to osteogenic differentiation of osteoblasts at day 10 under hypoxia was detected by qRT-PCR. Result sCompared with the control group, 200 nmol/mL CoCl₂ and 300 nmol/mL CoCl₂ had obvious inhibitory effect on osteoblast proliferation, and 300 nmol/mL CoCl₂ group had more obvious inhibitory effect on osteoblast proliferation. CoCl₂ promoted the apoptosis of osteoblasts in all groups, and 300 μmol/L was the most obvious. The apoptosis rate of osteoblasts increased with the increase of CoCl₂ concentration. Both 1 nmol/L and 5 nmol/L VitD₃ showed promoting effect on osteoblast proliferation under hypoxia condition, and the effect of 1 nmol/L VitD₃ was more significant. In terms of improving hypoxia and promoting osteoblast differentiation, 5 nmol/L VitD₃ showed superior promoting effect. Conclusion VitD₃ can promote the proliferation and differentiation of osteoblasts in rats under hypoxia.