Tumor progression has been recognized as the product of evolving crosstalk between cancer cells and the surrounding stromal cells. Cancer-associated orthotopic myofibroblasts may be linked to the progression of gastric carcinomas. To understand the significance of orthotopic myofibroblasts, we examined the effects of cancer-associated orthotopic myofibroblasts on the malignant phenotype of gastric cancer cells. Three human gastric cancer cell lines (OCUM-2MD3, OCUM-12, MKN-45) and four human gastric fibroblast cell lines (cancer-associated orthotopic fibroblast [CaF]-29, CaF-33, normal orthotopic fibroblast [NF]-29, NF-33) were used. The cancer-associated orthotopic fibroblast cell lines CaF-29 and CaF-33 were established from a tumoral gastric wall, and normal orthotopic fibroblast NF-29 and NF-33 were established from a non-tumoral gastric wall. Fibroblasts that were α-smooth muscle actin-positive were defined as myofibroblasts. We examined the effects of cancer-associated orthotopic myofibroblasts on the aggressiveness of gastric cancer cells by wound-healing assay, invasion assay, and RT-PCR. The ratios of myofibroblasts in CaF-29 (33%) and CaF-33 (46%) were significantly (< 0.001) greater than those in NF-29 (11%) or NF-33 (13%). Although all four orthotopic fibroblast lines increased the motility of gastric cancer cells, including migration and invasion ability, the motility-stimulating activity of cancer-associated fibroblasts (CaF-29 and CaF-33) was significantly higher than that of normal fibroblasts (NF-29 and NF-33). These motility-stimulating activities of cancer-associated orthotopic fibroblasts were downregulated by Smad2 siRNA treatment and anti-transforming growth factor-β neutralizing antibody. These findings suggest that cancer-associated orthotopic myofibroblasts may play an important role in the progression of gastric cancers and that transforming growth factor-β produced by myofibroblasts may be one of the factors associated with the aggressiveness of gastric carcinoma cells. (Cancer Sci 2012; 103: 797–805)