Background: Tracheal lesions are pathologies derived from the most diverse insults that can result in a fatal outcome. Despite the number of techniques designed for the treatment, a limiting factor is the extent of the extraction. Therefore, strategies with biomaterials can restructure tissues and maintain the organ’s functionality, like decellularized Wharton’s jelly (WJ) as a scaffold. The aim is to analyze the capacity of tracheal tissue regeneration after the implantation of decellularized WJ in rabbits submitted to a tracheal defect. Methods: An in vivo experimental study was undertaken using twenty rabbits separated into two groups (n = 10). Group 1 submitted to a tracheal defect, group 2 tracheal defect, and implantation of decellularized WJ. The analyses were performed 30 days after surgery through immunohistochemistry. Results: Inner tracheal area diameter (p = 0.643) didn’t show significance. Collagen type I, III, and Aggrecan highlighted no significant difference between the groups (both collagens with p = 0.445 and the Aggrecan p = 0.4). Conclusion: The scaffold appears to fit as a heterologous implant and did not trigger reactions such as rejection or extrusion of the material into the recipient. However, these results suggested that although the WJ matrix presents several characteristics as a biomaterial for tissue regeneration, it did not display histopathological benefits in trachea tissue regeneration.