Background This study aimed to investigate the effect of ovariectomy and vitamin D3 on bone microstructure; this effect was examined in three regions of interest at one femoral and two mandibular sampling sites bone in an ovariectomized mouse model. Methods Thirty-six week-old female mice were randomly divided into three groups: 10 subjects were given oral cholecalciferol (vitamin D3) daily for 6 weeks after undergoing bilateral ovariectomy (D3 group), while 10 ovariectomized subjects (OVX) and 10 subjects who underwent a sham operation (SHAM) received peanut oil daily during the investigation. After extermination, the left hemimandible and femur were removed and scanned by micro-CT. The bone micromorphology parameters were analyzed and the BMD was calculated. Results The bone volume fraction (BV/TV) was significantly lower in the trabecular bone of the mandibular condyle in the OVX group than in the SHAM and D3 groups. Also there was a significant difference between the SHAM and D3 groups. The specific bone surface (BS/BV) was significantly higher in the OVX and D3 groups than in the SHAM group. Trabecular thickness (Tb.Th) was significantly higher in the SHAM group, and the trabecular bone pattern factor (Tb.Pf) was significantly higher in the OVX group than in the other two groups. Bone mineral density (BMD) of the femur and the mandible was significantly lower in the OVX group than in the SHAM and D3 groups. Conclusions Our results show that ovariectomy causes a significantly weaker bone microstructure in the mandibular condyle, where the protective effect of vitamin D3 resulted in a partial resorption.