Communication between cancer cells and the microenvironment appears to be an important determinant of disease prognosis. However, the detailed mechanisms of the interactions between cancer cells and surrounding cells have yet to be clarified. Recent studies on cell fusion have indicated this interaction to be one of the driving forces in cancer progression. Fibroblasts constitute a significant component of the carcinoma stromal compartment. Many of these fibroblasts are thought to differentiate into myofibroblasts, which are characterized by a positive expression of α-smooth muscle actin. Expression of α-smooth muscle actin in osteosarcoma was evaluated, and was observed to be excessive in the multinucleated osteoclast-like giant cells in osteosarcoma tissue, indicating the possibility of cell fusion between cancer cells and myofibroblasts. In order to test the above hypothesis, we first transformed the primary mouse embryonic fibroblast cells into activated myofibroblast cells. Osteosarcoma cells were then co-cultured with mouse myofibroblast cells, and cell fusion was investigated using species-specific chromosomal markers. Expression of α-smooth muscle actin was successfully induced in primary mouse embryonic fibroblast cells. Cells fused spontaneously with a fusion rate of approximately 1-2% and fusion between more than two cells was also observed. Our study demonstrated that fusion between cancer cells and myofibroblasts may contribute to the observed multinucleated giant cells in osteosarcoma. We posit that cell fusion is a novel mechanism for the interaction between cancer cells and the microenvironment.