Astragaloside (AST) is derived from the Chinese herb Astragalus membranaceus, and studies have demonstrated that it promotes differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). To the best of our knowledge, however, the functions of the component AST-IV in osteogenesis have not previously been elucidated. The present study aimed to verify the effects of AST-IV in osteogenesis. First, the proliferation and differentiation status of human BMSCs incubated with AST-IV were analysed and compared with a control (no AST-IV treatment). In order to determine the involvement of the glycogen synthase kinase (GSK)3β signalling pathway in AST-IV, overexpression and inhibition of GSK3β was induced during incubation of BMSCs with AST-IV. In order to investigate how neuronal growth factor (NGF) contributes to BMSCs differentiation, BMSCs were co-incubated with an anti-NGF antibody and AST IV, and then levels of osteogenesis markers were assessed. The results demonstrated for the first time that AST-IV contributed to BMSCs differentiation. Furthermore, the GSK3β/β-catenin signalling pathway was revealed to be involved in AST-IV-induced osteogenesis; moreover, AST-IV accelerated differentiation by enhancing the expression levels of NGF. In summary, the present study demonstrated that AST-IV promotes BMSCs differentiation, thus providing a potential target for the treatment of osteoporosis.