To prepare scaffolds for heart valve tissue engineering, porcine heart valves were treated with varied concentrations of trypsin for 32, 56, 80 and 104 h or followed with DNase. And then the structure of acellular valves was observed under light microscope, scanning and transmission electron microscope. Porcine endothelial cells, human endothelial cells, and canine myofibroblasts were reseeded onto the acellularized porcine heart valve scaffolds once a day for 3 days. The valves were analyzed by immunohistochemical staining and electron microscopy. Results show that all endothelial cells and the majority of interstitial cells were removed from the heart valves after digestion with trypsin for 104 h, and the collagen fiber structure remains intact, but the space between collagen fibers increased slightly. Incubation with trypsin for 80 h and then with DNase almost removed all cells, and the collagen fiber structure and the space between the fibers remain intact. After reseeding, human endothelial cells almost fully cover the valve scaffold surface as shown by H-E staining and platelet endothelial cell adhesion molecules (PECAM-1) staining. Xenogeneic porcine endothelial cells also adhered to and grew on the scaffolds. As shown by H-E staining and actin staining, canine myofibroblasts not only adhered to the surface of valve scaffold but also migrated to the inner part of matrix after one week culture. These results suggest that the digestion of porcine heart valves with trypsin combining with DNase is a suitable method to remove cells. The acellular porcine heart valve scaffolds have a quite favorable biocompatibility with human and porcine endothelial cells as well as canine myofibroblasts.