Antimetabolite Methotrexate (MTX) is commonly used in childhood oncology. As a dihydrofolate reductase inhibitor it exerts its action through the reduction of cellular folate, thus its intensive use is associated with damage to soft tissues, bone marrow, and bone. In the clinic, MTX is administered with folinic acid (FA) supplementation to alleviate some of this soft tissue damage. However, whether and how FA alleviates damage to the bone and bone marrow requires further investigation. As the Wnt/β-catenin signalling pathway is critical for commitment and differentiation of mesenchymal stem cells down the osteogenic or adipogenic lineage, its deregulation has been found associated with increased marrow adiposity following MTX treatment. In order to elucidate whether FA supplementation prevents MTX-induced bone marrow adiposity by regulating Wnt/β-catenin signalling, young rats were given saline or 0.75mg/kg MTX once daily for 5 days, receiving saline or 0.75mg/kg FA 6h after MTX. FA rescue alleviated the MTX-induced bone marrow adiposity, as well as inducing up-regulation of Wnt10b mRNA and β-catenin protein expression in the bone. Furthermore, FA blocked up-regulation of the secreted Wnt antagonist sFRP-1 mRNA expression. Moreover, secreted sFRP-1 protein in the bone marrow and its expression by osteoblasts and adipocytes was found increased following MTX treatment. This potentially indicates that sFRP-1 is a major regulator of defective Wnt/β-catenin signalling following MTX treatment. This study provides evidence that folate depletion caused by MTX chemotherapy results in increased bone marrow adiposity, and that FA rescue alleviates these defects by up-regulating Wnt/β-catenin signalling in the bone.