In this work, a new method for producing acellular dermis (ADM), a natural scaffold used for dermal replacement, from porcine skin was developed. Fresh porcine skin from local slaughterhouse was dehaired by sodium sulphide following by epidermis removal using glycerol. After fat removal by chloroform/methanol (2/1 v/v) solvent, cellular components were removed using enzymatic treatment incorporated with a periodic pressurized technique. The effects of enzyme type (trypsin and dispase II) and periodic pressurized conditions on the efficiency of cell removal were investigated. When periodic pressure was applied, enzymatic treatment time could be shorten since the enzyme solution was able to penetrate into tight dermis. As a result, cells could be easily removed from porcine skin as noticed quantitatively by DNA assay and qualitatively by H&E staining. When enzyme refreshment was introduced into the decellularized process, the percentage of cell removal was further enhanced. This ensured that no inhibitions effect from the removed cells on enzyme-substrate interaction. Moreover, short-time enzymatic treatment with periodic pressurized technique could prevent the disruption of dermal structure, as observed by SEM. Dispase II can be used to remove cell better than trypsin in the periodic pressurized technique. However, in vivo study indicated that numerous fibroblast from the host tissue infiltrated into ADM prepared using both enzymes. Neo-collagen and neo-capillaries were produced in both implanted ADMs. The result elucidated that the use of periodic pressurized technique with enzymatic treatment has a high potential to be a new method to produce ADM for skin tissue engineering.