Abstract: To alleviate the mass transport limit during ex vivo tissue engineering, a perfusion culture bioreactor was developed for fabricating dermal substitutes, and the effects of medium perfusion rate on cell proliferation, distribution and extracellular matrix (ECM) deposition was investigated. PET scaffolds seeded with human dermal fibroblasts by perfusion were cultivated for 18 d at flow rates of 0.02, 0.2, 0.8 cm/min, respectively. It was found that perfusion culture favored cell proliferation, aerobic metabolism as well as a uniform distribution of cells and ECM in 3D engineered constructs. In addition, when compared to that at 0.02 cm/min and static culture, perfusion at 0.2 cm/min and 0.8 cm/min significantly stimulated glucose consumption and collagen production. However, a greater leakage of collagen content in culture medium was also observed when perfused at 0.2 cm/min and 0.8 cm/min. These data suggested that perfusion culture is a promising method for dermal tissue engineering.