Most of the clinically available decellularized tissues are preserved in a freeze-dried state. Freeze-dried (FD) tissues can be preserved for long term, although a rehydration process is necessary before use. Currently, an immersion method is most commonly used in clinical procedures, but it is difficult for complicated and thick structure tissue rehydration. In this study, we tried to apply a vacuum pressure impregnation (VPI) technique for FD tissue rehydration. The water content of decellularized tissues can reach the water content of native tissues within 30 min using VPI, whereas it took 6 h to reach the same water content using the immersion method. Furthermore, heparin rehydrated aortas by VPI had more heparin release at each time point and therefore appeared more anticoagulant activity. We found that the VPI treatment promotes solution infiltration into materials, achieves complete rehydration of the decellularized tissues, and deep infiltration of heparin into the decellularized tissues, suggesting that VPI treatment could be applied as a rehydration method for biological materials.