PURPOSE: Abnormal growth of vertebral body growth plate (VBGP) is considered as one of the etiologic factors in the adolescent idiopathic scoliosis (AIS). It was well-known that melatonin was correlated with the emergence and development of AIS. This study aimed to investigate the effect of melatonin on rat VBGP chondrocytes in vitro. METHODS: Chondrocytes were isolated from rat VBGP, and treated with or without melatonin. Cell proliferation was measured by the Alamar Blue assay. Gene expression of collagen type II and aggrecan were evaluated by real-time PCR. Expression of the melatonin receptors (MT1, MT2), proliferating cell nuclear antigen (PCNA, a cell proliferation marker), Sox9 (a chondrocytic differentiation marker) and Smad4 (a common mediator in regulating the proliferation and differentiation of chondrocytes) were detected by Western blotting. RESULTS: Expression of melatonin receptors (MT1, MT2) were detected in the rat VBGP chondrocytes. Melatonin, at 10 and 100 µg/mL concentration, significantly inhibited the proliferation of VBGP-chondrocytes and the gene expression of collagen type II and aggrecan, and down-regulated the protein expression of PCNA, Sox9 and Smad4. In addition, the inhibitory effect of melatonin was reversed by luzindole, a melatonin receptor antagonist. CONCLUSIONS: These results suggest that melatonin at high concentrations can inhibit the proliferation and differentiation of VBGP chondrocytes, which might give some new insight into the pathogenic mechanism of AIS.