Yes-associated protein 1 (YAP1) is a candidate oncogene that is involved in tumorigenesis and progression of many malignant tumors. Recently, many studies have revealed that YAP1 is highly expressed in human osteosarcoma. To investigate the role of YAP1 in osteosarcoma tumorigenesis, the expression of YAP1 in the osteosarcoma cell lines (MG-63 and HOS) was knocked down by small hairpin RNA (shRNA), and the cell proliferation and colony formation assay showed that knockdown of YAP1 significantly suppressed the cell proliferation and colony formation of osteosarcoma cells. Subsequently, cell cycle distribution was analyzed by flow cytometry, and the results showed an accumulation of YAP1-knockdown cells in the G0/G1 phase, suggesting that YAP1 knockdown results in the arrest of cell cycle progression. Additionally, the knockdown of YAP1 also inhibited the tumorsphere formation in vitro and the growth of xenograft tumors in vivo. Therefore, these data suggest that YAP1 knockdown inhibits the proliferation of osteosarcoma cells. However, the mechanism of action was unclear. Further investigation showed that in the YAP1-knockdown MG-63 and HOS cells, the level of cylinD1 and c-myc expression, target genes of the Wnt signaling pathway and TOP-Flash reporter activity were all significantly decreased, which indicated that the inhibitory effect of YAP1 knockdown on osteosarcoma might be associated with the Wnt signaling pathway. Taken together, our results demonstrated that YAP1 is an important regulator of osteosarcoma tumorigenesis and knockdown of YAP1 would be a novel therapeutic strategy for osteosarcoma.