Autologous chondrocyte transplantation (ACT) is a promising method to treat chondral and osteochondral defects. This study introduced a modified method for cell culture in ACT. Porcine chondrocytes were cultured for 3 weeks under low hydrostatic pressure at 250 Pa. The results showed that the dry weight of the cartilage-like membrane in the loading group was 3.0 times more than the control group (no loading) (p < 0.01), and cell numbers were significantly increased by 3.1 times (p < 0.01) after a 3-week culture. Compared with the fresh tissue sample, the mRNA expression of collagen II was not statistically different and the mRNA of aggrecan was only slightly decreased by 19%. These data suggest that the hydrostatic pressure at this level significantly increased the cell numbers and biosynthesis of cultured chondrocytes.