The purpose of this study is to evaluate the feasibility of human amniotic membrane (HAM) as a chondrocyte carrier by assessing cell proliferation and maintenance of phenotype in vitro and cartilage regeneration in vivo. Intact HAM was treated with 0.1% trypsin-ethylenediaminetetraacetic acid (EDTA) for 15 min and the epithelial cells removed to make a denuded HAM. Rabbit articular chondrocytes were then seeded on three different HAM substrates: the epithelial side of intact HAM (IHE), basement side of denuded HAM (DHB), and stromal side of denuded HAM (DHS). These cell-substrate specimens were cultured for up to 4 weeks, and cell proliferation rate and phenotypic stability were examined at weeks 1 and 4. While chondrocytes grew in monolayer fashion on the surface of IHE and DHB substrates, the cells seeded in DHS penetrated and spread into the whole thickness of the stromal layer. The proliferating activity of chondrocytes in DHB was continuously up-regulated. A similar proliferating activity was observed in DHS in the first week, which remained stable for up to 4 weeks. The expression of type II collagen gradually increased with time in the DHS group, while it gradually decreased in the DHB group or was not detected at all in the IHE group. These results suggested that denuded HAM was able to support chondrocyte proliferation and maintenance of phenotype in vitro, seemingly more favorable when DHS was used. Based on this data, the DHS with chondrocytes was used to cover rabbit osteochondral defect with the stromal side facing in. The defect area was successfully regenerated with hyaline cartilage in the Safranin-O stain and International Cartilage Repair Society (ICRS) scoring after 8 weeks of implantation. In conclusion, our findings suggest that denuded HAM could be one of the ideal cell carrier matrices for cartilage regeneration.