Background: The development of innovative therapies for bone regeneration requires the use of advanced site-specific bone defect small-animal models. The achievement of proper fixation with a murine model is challenging due to the small dimensions of the murine femur. The aim of this investigation was to find the optimal defect size for a murine critical-size bone defect model using external fixation method. Methods: An external fixation device was attached to the right femur of 30 mice. Femoral bone defects of 1 mm (n = 10), 2 mm (n = 10), and 3 mm (n = 10) were created. Wounds were closed without any additional treatment. To investigate bone healing during the 12-wk observation period, x-ray analysis, histomorphology, immunohistochemistry, and μCT scans were performed. Results: MicroCT analyses after 12 wk showed that 3/8 1-mm defects, 5/8 2-mm defects, and 8/8 3-mm defects remained as nonunions. The defect volumes were 0.36 ± 0.42 mm³ (1-mm group), 1.40 ± 0.88 mm ³ (2-mm group), and 2.88 ± 0.28 mm³ (3-mm group; P < 0.001, between all groups). Conclusion: Using external fixation, a defect size of 3 mm is necessary to reliably create a persisting femoral bone defect in nude mice.