Background: A preclinical study was conducted for evaluating a valved conduit manufactured with a glutaraldehyde (GA)-fixed bovine pericardium treated using an anticalcification protocol. Methods: Bovine pericardia were decellularized, fixed with GA in an organic solvent, and detoxified. We prepared a valved conduit using these bovine pericardia and a specially designed mold. The valved conduit was placed under in vitro circulation by using a mock circulation model, and the durability under mechanical stress was evaluated for 2 months. The valved conduit was implanted into the right ventricular outflow tract of a goat, and the hemodynamic, radiologic, histopathologic, and biochemical results were obtained for 6 months after the implantation. Results: The in vitro mock circulation demonstrated that valve motion was good and that the valved conduit had good gross and microscopic findings. The evaluation of echocardiography and cardiac catheterization demonstrated the good hemodynamic status and function of the pulmonary xenograft valve 6 months after the implantation. According to specimen radiography and a histopathologic examination, the durability of the xenografts was well preserved without calcification at 6 months after the implantation. The calcium and inorganic phosphorus concentrations of the explanted xenografts were low at 6 months after the implantation. Conclusion: This study demonstrated that our synergistic employment of multiple anticalcification therapies has promising safety and efficacy in the future clinical study.