Methotrexate (MTX) is commonly used in cancer chemotherapy to treat childhood leukaemia and osteosarcoma. Although the application of MTX chemotherapy improves the population of survivors, the prevalence of chronic bone-related complications has increased. Reduced bone formation (osteogenesis) and increased marrow fat formation (adipogenesis) have been observed through a "switch-like" change in commitment of bone marrow stromal cells (BMSCs) following MTX treatment. However, the underlying molecular mechanisms of this bone/fat switch are not fully elucidated. MicroRNAs participate in regulating BMSC differentiation by targeting the 3' untranslated region of osteogenic/adipogenic related genes. Here, in bone samples from MTX-treated rats, we found some differentially expressed microRNAs by microRNA array and RT-PCR. In addition, we found some microRNAs which have a negative correlation with expression of TGF-β signalling molecule Smad2 or Wnt antagonist sFRP-1. In vitro cell models were applied to validate the effects of microRNA agomir and antagonist delivery. Results suggest that overexpression of these microRNAs (microRNA-6315 & microRNA-542-3p) enhanced osteogenic differentiation, while their inhibition had the opposite effects. In addition, we found that the expression of these microRNAs was positively correlated with bone formation marker genes RUNX2, ALP, OCN and OSX. Furthermore, target prediction and dual-luciferase assays identified Smad2 and sFRP-1 as the direct targets for these microRNAs. Since Smad2 and sFRP-1 are the essential regulators involved in TGF-β and Wnt/β-catenin signalling pathways which are associated with osteogenesis and adipogenesis in BMSCs, the microRNAs we identified are likely to regulate osteogenesis and adipogenesis and the bone/fat switch after MTX treatment via the TGF-β and Wnt/β-catenin signalling pathways.