OBJECTIVES Graphene oxide (GO), the derivative of graphene with unique properties, has attracted much attention for applications in dental implants. The aim of this study was, by two biomimetic cell culture methods, to investigate the quantitative relationship between the concentration of pristine GO nanosheets and their cellular behaviours towards bone marrow-derived mesenchymal stem cells (BMSCs). MATERIALS AND METHODS The cells were firstly characterized according to their morphology, self-renewal capabilities and multipotency. Subsequently, adhesion density, proliferation, alkaline phosphatase activity and mineralization of BMSCs treated with various concentrations of GO were analysed. In addition, osteogenic-related proteins were measured for further verification of the GO-induced osteogenic differentiation. RESULTS Pristine GO nanosheets inhibited the proliferation of BMSCs at a high concentration of 10 μg/mL during the first 3 days with two seeding methods and facilitated proliferation of BMSCs at a low concentration of 0.1 μg/mL after 5 days with a sequential-seeding method compared to a co-seeding method. Analogously, osteogenic differentiation was promoted when BMSCs were treated with 0.1 μg/mL of GO. Both the proliferation and differentiation showed concentration-dependent behaviour. Interestingly, Wnt/β-catenin signalling pathway appeared to be involved in osteogenic differentiation induced by pristine GO nanosheets. CONCLUSIONS Pristine GO nanosheets at a concentration of 0.1 μg/mL provide benefits to promote BMSCs proliferation and osteogenesis under a sequential-seeding method, contributing to the use of GO for dental implantation.