The aim of this study was to fabricate and characterize acellular porcine pericardium that can be used to patch cardiovascular repair. Porcine pericardial tissues were treated with 10 mM Tris-HCl and sodium dodecyl sulfate (SDS) at different concentrations and time intervals. The optimal decellularization protocol was determined according to elimination of cellular components and DNA via histology and DNA quantification, respectively. There were no significant changes in stress and fracture strain after decellularization. Liquid extracts from the decellularized pericardium caused no cytotoxicity towards human fibroblasts, were capable of supporting an appropriate attachment of human endothelial progenitor cells, and did not cause a local inflammatory effect after 30 days of transplantation in mice.