Objective To investigate the effects of vitamin D ₃ (VitD ₃) 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 CoCl ₂), CoCl ₂ group (300 nmol/mL CoCl ₂) and VitD ₃ group (300 nmol/mL CoCl ₂ + 1 nmol/L VitD ₃). VitD ₃ group 2(300 nmol/mL CoCl ₂ + 5 nmol/L VitD ₃). 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 CoCl ₂ and 300 nmol/mL CoCl ₂ had obvious inhibitory effect on osteoblast proliferation, and 300 nmol/mL CoCl ₂ group had more obvious inhibitory effect on osteoblast proliferation. CoCl ₂ 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 CoCl ₂ concentration. Both 1 nmol/L and 5 nmol/L VitD ₃ showed promoting effect on osteoblast proliferation under hypoxia condition, and the effect of 1 nmol/L VitD ₃ was more significant. In terms of improving hypoxia and promoting osteoblast differentiation, 5 nmol/L VitD ₃ showed superior promoting effect. Conclusion VitD ₃ can promote the proliferation and differentiation of osteoblasts in rats under hypoxia.