The aim of the present study was to investigate the influence of a decellularization protocol on the structure and the mechanical behavior of small-diameter (<6 mm) tibial calf arteries and veins. Calf vessels were decellularized by a detergent-enzymatic method (DEM), partially hydrolyzed with trypsin and subsequently cross-linked using poly(ethylene glycol) diglycidyl ether. Our results showed that i) the DEM can be considered a simple and valuable procedure for the preparation of complete acellular arteries and veins able to preserve a high degree of collagen and elastic fibers, and ii) poly(ethylene glycol) diglycidyl ether cross-linking treatment provides appropriate mechanical reinforcement of blood vessels. Histologically, the decellularized vessels were obtained employing the detergent-enzymatic procedure and their native extracellular matrix histoarchitecture and components remained well preserved. Moreover, the decellularization protocol can be considered an effective method to remove HLA class I antigen expression from small-diameter tibial calf arteries and veins. Cytocompatibility of decellularized cross-linked vessels was evaluated by endothelial and smooth muscle cell seeding on luminal and adventitial vessel surfaces, respectively.