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Introduction: Decellularized larynges could be used as scaffolds to regenerate the larynx. The purpose of this study was to establish a perfusion decellularization protocol to produce a 3-dimensional whole laryngeal extracellular matrix (ECM) scaffold in a rabbit model. Methods: The larynges of 20 rabbits assigned to the study group were harvested and decellularized using a perfusion decellularization protocol, while the larynges of 10 rabbits in the control group were harvested and untreated. Macroscopic and microscopic morphological analyses, a molecular analysis, a cellular content analysis, and scanning electron microscopy were performed. Results: A histological analysis showed the absence of cellular components, the presence of the ECM, and an intact cartilage structure filled with chondrocytes. The mean total DNA amounts of the native larynx, decellularized larynx, and decellularized cartilage-free larynx were 1,826.40, 434.70, and 41.40 μg/µL, respectively; those for the decellularized larynx and decellularized cartilage-free larynx were significantly lower (p < 0.001 and p < 0.001, respectively). The total amount of DNA in the decellularized sample was significantly lower compared to that in the native sample, at 57.2% in cartilage (p < 0.001), 2.4% in the thyroid gland (p < 0.001), 2.7% in muscle (p < 0.001), 1.6% in vessels (p < 0.001), and 4.8% in the vocal cords (p < 0.001). Conclusion: Our perfusion decellularization protocol is feasible and reproducible to produce a 3-dimensional whole laryngeal ECM scaffold in a rabbit. [ABSTRACT FROM AUTHOR]