Adolescent idiopathic scoliosis (AIS) is a complex, three-dimensional deformity of the spine that commonly occurs in pubescent girls. Decreased osteogenic differentiation and aberrant melatonin signalling have been demonstrated in mesenchymal stem cells (MSCs) from AIS patients and are implicated in the pathogenesis of AIS. However, the molecular mechanisms underlying these abnormal cellular features remain largely unknown. Our previous work comparing gene expression profiles between MSCs from AIS patients and healthy controls identified 1027 differentially expressed genes. In the present study, we focused on one of the most downregulated genes, SPRY4, in the MAPK signalling pathway and examined its role in osteogenic differentiation. We found that SPRY4 is markedly downregulated in AIS MSCs. Knockdown of SPRY4 impaired differentiation of healthy MSCs to osteoblasts, while SPRY4 overexpression in AIS MSCs enhanced osteogenic differentiation. Furthermore, melatonin treatment boosted osteogenic differentiation, whereas SPRY4 ablation ablated the promotional effects of melatonin. Moreover, SPRY4 was upregulated by melatonin exposure and contributed to osteogenic differentiation and melatonin response in a MEK-ERK1/2 dependent manner. Thus, loss of SPRY4 in bone marrow derived-MSCs results in reduced osteogenic differentiation, and these defects are further aggravated under the influence of melatonin. Our findings provide new insights for understanding the role of melatonin in AIS aetiology and highlight the importance of MSCs in AIS pathogenesis.