BACKGROUND: A tissue engineering technique based on use of the extracellular matrix (ECM) as a scaffold shows great potential for preparing small-caliber vascular grafts. Decellularization protocols are still not standardized for bioengineering. The effects of freeze-thaw cycles used for decellularization are unknown. OBJECTIVE: To evaluate the effects of freeze-thaw cycles on porcine carotid arteries during decellularization and to develop a promising protocol for preparing ECM scaffolds. METHODS: Porcine carotid arteries were decellularized with freeze-thaw cycles followed by three different chemical protocols. Histological analysis, scanning electron microscopy, mechanical tests and pore size measurement were performed to assess their effects on the ECM. RESULTS: The composition, structure, and mechanical properties were not significantly changed after freeze-thaw cycles, with the exception of endothelial cells loss. Freeze-thaw led to a porous structure within arteries. The use of Triton X-100 followed by sodium dodecyl sulfate (SDS) resulted in ECM scaffolds with well-preserved composition, structure, and mechanical properties, as well as with adequate porosity. CONCLUSIONS: As the initial step for decellularization, freeze-thaw had little impact on arteries. Decellularized porcine carotid arteries, prepared using freeze-thaw cycles followed by treatment with Triton X-100 and SDS, may serve as a promising biological scaffold as a tissue-engineered vascular graft.