•Any decellularization impairs tracheal cartilage, trachealis, and connective tissue.•Treatment causes loss of Coll-II and GAGs, leading to collapse of cartilage rings.•Coll-III is lost with any treatment whereas Col-I becomes disorganized and degrades.•Trachealis muscle becomes more compliant with loss of contractile SMCs.•Axial mechanics are governed by collagen structure and longitudinal elastin fibers. Tracheal replacement using tissue engineering technologies offers great potential to improve previously intractable clinical interventions, and interest in this area has increased in recent years. Many engineered airway constructs currently rely on decellularized native tracheas to serve as the scaffold for tissue repair. Yet, mechanical failure leading to airway narrowing and collapse remains a major cause of morbidity and mortality following clinical implantation of decellularized tracheal grafts. To understand better the factors contributing to mechanical failure in vivo, we characterized the histo-mechanical properties of tracheas following two different decellularization protocols, including one that has been used clinically. All decellularized tracheas deviated from native mechanical behavior, which may provide insights into observed in vivo graft failures. We further analyzed protein content by western blot and analyzed microstructure by histological staining and found that the specific method of decellularization resulted in significant differences in the depletion of proteoglycans and degradation of collagens I, II, III, and elastin. Taken together, this work demonstrates that the heterogeneous architecture and mechanical behavior of the trachea is severely compromised by decellularization. Such structural deterioration may contribute to graft failure clinically and limit the potential of decellularized native tracheas as viable long-term orthotopic airway replacements. Display omitted