Objective To compare the effect of different defect diameters on healing in the middle 1/3 tibia monolayer cortical bone defect mouse model so as to establish an animal model for bone tissue engineering study, mechanism study on bone defect repair, and gene therapy research. Methods Ten 8-week-old C57BL/6J mice, weighing (20 ± 2) g, were randomly divided into 2 groups, 5 mice in each group. The middle 1/3 tibiae monolayer cortical bone defect model of 0.8 mm (group A) or 1.0 mm (group B) in diameter was established with burr drill. At 7, 21, and 28 days after modeling, the molybdenum target X-ray radiography was used to observe the defect repair; at 28 days, Micro CT and three-dimensional imaging were used to evaluate bone defect repair, and tibia specimens were harvested for HE staining. Results At 7 days after modeling, tibia fracture occurred in 5 mice in group B, no fracture in group A. X-ray films, Micro CT scan, and HE staining showed bony union in group A at 28 days. The quantitative analysis of trabecular bone by Micro CT showed that trabecular number, connectivity density, and bone volume in group A were significantly greater than those in group B (P lt; 0.05), mean of segmented region—mean 2 was significantly less than that in group B (P lt; 0.05), but no significant difference was found in trabecular separation and trabecular thickness between 2 groups (P gt; 0.05). Conclusion The middle 1/3 tibia monolayer cortical bone defect mouse model of 0.8 mm in diameter is the ideal animal model for study repair mechanism of tibia defect or bone tissue engineering.