Objective To investigate the effect of 1,25(OH)2VD3 on differentiation of embryonic stem cells (ESCs) into osteoblasts. Methods Osteoblasts were isolated and cultured from calvarium of 2-day-old Kunming white mice, embryoid bodies (EBs) were prepared with modified zur Nieden method. EBs were divided into 4 groups according to different mediums: group A, as the control group, in which EBs medium contained no leukemia inhibitory factor; group B, in which EBs medium contained supplements of Vitamin C (VC, 50 μg/mL) and β-glycerophosphate (β-GP, 50 mmol/L); group C, inwhich EBs medium was the same as that of group B and 5 × 104 osteoblasts of 3rd passage were seeded into each well; group D, in which the medium contained supplements of VC (50 μg/mL), β-GP (50 mmol/L) and 1,25(OH)2VD(4 × 10-9 mol/L), and 5 × 104 osteoblasts of 3rd passage were seeded into each well. The ALP activity was determined by ALP reagent kit every 5 days. The RQ-PCR was performed to measure the mRNA expressions of osteocalcin (OCN). Al izarin red S staining was performed to count the bone nodules. Results The expression of ALP witnessed no obvious change in each group within 5 days after adherence of EBs, but increased gradually after 5 days. The expression of ALP in group D reached the peak at 20 days. Red nodules with clear outl ine and different sizes were evident by microscope. Al izarin red S staining testified the number of bone noudles in groups A, B, C and D was 20 ± 8, 18 ± 5, 31 ± 1 and 50 ± 1, respectively, indicating significant differences between groups C, D and groups A, B (P lt; 0.05), no significant difference between group A and group B (P gt; 0.05), and a significant difference between group C and group D (P lt; 0.05). The result of RQ-PCR showed that the mRNA expressions of OCN in groups A, B, C and D was 10.18 ± 1.17, 20.29 ± 1.03, 18.84 ± 4.07 and 32.15 ± 5.23, respectively, indicating significant differences between groups C, D and groups A, B (P lt; 0.05), no significant difference between group A and group B (P gt; 0.05), and a significant difference between group C and group D (P lt; 0.05). Conclusion The combined action of 1,25(OH)2VD(4 × 10-9 mol/L), VC, and β-GP can effectively promote the differentiation of the ESCs-derived osteoblasts.