To develop a minimally invasive preventive measure for early osteoarthritis, the effect of melatonin on cartilage matrix synthesis of articular chondrocytes was evaluated in vitro in a pellet culture system. The chondrogenic markers were assessed using histology, TaqMan polymerase chain reaction, and western blot. Our results show that melatonin treatment yielded chondrocyte-pellets with a higher expression of chondrogenic markers consisting of collagen II, Sox 9, and aggrecan at both the mRNA and protein levels. A hypertrophic marker, collagen X, remained low. Moreover, up-regulation of internal transforming growth factor beta1 (TGF-beta1) expression was observed in the melatonin-treated cells. Our data indicate, for the first time, that the administration of melatonin enhances cartilage matrix synthesis of articular chondrocytes in a serum-containing pellet culture system, likely through the TGF-beta signal pathway. Melatonin may prove to be a highly valuable addition to current therapeutic models for degenerative cartilage repair.