Bointerface mediated physical cues have been found to be able to guide stem cells to differentiate into specific cell lineages. In this study, nanostructured MoS2 biointerface was fabricated by assembling few-layer MoS2 nanoflakes onto substrate using a facile hydrothermal method. Its effects on proliferation, adhesion and osteogenic differentiation of rat bone marrow mesenchymal stem cells (MSCs) were compared with a flat substrate. The live–dead staining and cell count kit-8 (CCK-8) were used to evaluate the biocompatibility. Focal adhesion formation was used to explore the interaction between MSCs and the nanostructured MoS2. Alkaline phosphatase (ALP) activity, alizarin red S staining, immunofluorescent staining and real quantitative polymerase chain reaction (RT-qPCR) were performed to assess the osteogenic differentiation of MSCs. The results showed that the nanoporous structured MoS2 interface can not only promote the MSCs attachment and spreading, but also accelerate the MSCs osteogenesis. Our results can provide insight into the design considerations of the biointerface to control the stem cell fate.