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Importance: Advances in tissue engineering offer potential alternatives to current mandibular reconstructive techniques; however, before clinical translation of this technology, a relevant animal model must be used to validate possible interventions., Objective: To establish the critical-sized segmental mandibular defect that does not heal spontaneously in the rat mandible., Design and Setting: Prospective study of mandibular defect healing in 29 Sprague-Dawley rats in an animal laboratory., Interventions: The rats underwent creation of 1 of 4 segmental mandibular defects measuring 0, 1, 3, and 5 mm. All mandibular wounds were internally fixated with 1-mm microplates and screws and allowed to heal for 12 weeks, after which the animals were killed humanely., Main Outcomes and Measures: Analysis with micro-computed tomography of bony union and formation graded on semiquantitative scales., Results: Seven animals were included in each experimental group. No 5-mm segmental defects successfully developed bony union, whereas all 0- and 1-mm defects had continuous bony growth across the original defect on micro-computed tomography. Three of the 3-mm defects had bony continuity, and 3 had no healing of the bony wound. Bone union scores were significantly lower for the 5-mm defects compared with the 0-, 1-, and 3-mm defects (P < .01)., Conclusions and Relevance: The rat segmental mandible model cannot heal a 5-mm segmental mandibular defect. Successful healing of 0-, 1-, and 3-mm defects confirms adequate stabilization of bony wounds with internal fixation with 1-mm microplates. The rat segmental mandibular critical-sized defect provides a clinically relevant testing ground for translatable mandibular tissue engineering efforts.