Osteosarcoma (OS) is one of the most common malignant bone tumors in early adolescence. Multi-drug chemotherapy has greatly increased the five year survival rate from 20% to 70%. However, the rate has been staggering for 30 years and the prognosis is particularly poor for patients with recurrence and metastasis. Our study aimed to investigate the role of Wnt-beta-catenin, Notch and Hedgehog pathway in OS development because all these pathways are involved in skeletal development, tumorigenesis and chemoresistance. Our results showed that the major components in Wnt-beta-catenin pathway, e.g. Wnt3a, beta-catenin and Lef1, were consistently upregulated in human osteosarcoma cell line Saos2 cells compared to human fetal osteoblasts (hFOB), whereas the changes in the expression levels of Notch and Hh signaling molecules were not consistent. Knocking down beta-catenin increased the Saos2 sensitivity to methotrexate (MTX) induced cell death. Consistently, the expression level of beta-catenin protein correlated with the invasiveness of OS, as evidenced by more intensive beta-catenin immunoreactivity in higher grade OS samples. Chemical inhibition of the Wnt-beta-catenin signaling enhanced MTX mediated death of Saos2 cells. A synergistic effect with MIX was observed when both inhibitors for Wnt-beta-catenin and Notch pathways were simultaneously used, while the addition of the Hh inhibitor did not further improve the efficacy. Our findings provide some novel insight to OS pathogenesis and lay a foundation for future application of Wnt-beta-catenin and Notch inhibitors together with the currently used chemotherapeutic drugs to improve the outcome of OS treatment. Published by Elsevier Inc.