INTRODUCTION AND OBJECTIVES: Colorectal cancer (CRC) is described as the second leading cause of cancer death worldwide, which is justified by its high rate of recurrence and metastatic capacity. Radiotherapy is the most effective form of cytotoxic treatment available in the treatment of solid cancers, and it can modulate the tumor microenvironment. Studies have shown that radiation therapy can activate fibroblasts, turning them into Cancer-Associated Fibroblasts (CAFs), which appear to be resistant to radiation. In addition, it has been seen that CAFs are able to modify the secretion of extracellular matrix (ECM) proteins, increasing the deposition of type I collagen, fibronectin, laminin, proteoglycans and tenascin-C, being able to trigger epithelial-mesenchymal transition (EMT) in cancer cells. Thus, we hypothesize that irradiated fibroblast-derived extracellular matrix could modify the behavior of colorectal cancer cells (CCR), through the activation of pathways that lead to EMT. MATERIAL AND METHODS: Initially, fibroblasts FGH were irradiated with 25 Gy, 5Gy for 5 days, and in the last day we stimulated two groups with TGF-β for 72hrs.Then cell viability was evaluated through MTT assay to access the effect of irradiation on cell growth. Next, we verified whether the irradiation stimulus was able to activate fibroblasts into a CAF phenotype. The molecular markers characteristic of the CAF phenotype was evaluated through immunoblotting and immunofluorescence. RESULTS AND CONCLUSION: Our results suggests that the irradiation didn’t affect the cell viability of fibroblasts, as all groups maintained a similar number of cells. We also observed a significant increase in the expression of α-SMA, a classic marker of CAF activation, especially in the fibroblasts that were irradiated and stimulated with TGF- β, which was corroborated by the immunofluorescence assay. Furthermore, we observed a tendency of an increase in SNAIL-1 expression in the same group treated with irradiation and TGF-β. We did not observe significant differences in the protein expression of vimentin, SNAIL and FAP. These data suggests that our protocol of irradiation induced an activated phenotype in fibroblasts, which could modify its own extracellular matrix and trigger EMT, playing an important role in tumor progression.