Background: The optimal management of benign tracheal stricture remains surgical resection. Resection is not always an option because of the challenges posed by anastomotic tension and a tenuous blood supply. Regenerative medicine approaches, such as extracellular matrix (ECM) scaffold technology, may alleviate some of the limitations to tracheal replacement. ECM scaffolds facilitate site-specific tissue remodeling when used to reconstruct a variety of soft-tissue structures. Methods: A 1-cm wide × 2-cm-long defect was created in the ventral trachea of 15 dogs and repaired with one of three acellular biologic scaffolds: urinary bladder matrix (UBM), UBM crosslinked with carbodiimide (UBMC), and decellularized tracheal matrix (DTM). The grafts were evaluated periodically using bronchoscopy and by macroscopic and microscopic morphologic examination at either 2 months or 6 months. Results: The UBM, UBMC, and DTM groups showed no evidence of stenosis or tracheomalacia. The UBM, UBMC, and DTM groups all showed deposition of organized collagenous tissue at the site of scaffold placement and an intact epithelial layer. Scattered areas of mucociliary differentiation were present at the edges of the graft site. There was no evidence cartilage observed within the remodeled tissue at 6 months. Conclusions: ECM scaffolds promote healing of significantly sized tracheal defects without stricture and with some, but not all, of the necessary structures required for tracheal reconstruction, including complete coverage with ciliated epithelium and dense organized collagenous tissue.