AbstractColorectal Cancer (CRC) therapy confronts challenges as chemoresistance and side effects. Therefore, drugs with antitumor properties that downmodulate aggressiveness mediators are required. Studies have shown the relevance of Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP), Protein Tyrosine Phosphatase 1B (PTP1B), and Transforming Growth Factor β (TGFβ) in mediating proliferation, chemoresistance, and metastasis. In this study, we aimed to investigate the responsiveness of colorectal cancer lines (HT29 and HCT116) towards Vemurafenib and whether this treatment could modulate these aggressiveness mediators. Cytotoxicity Assays (MTT and Trypan Exclusion Test) were performed to evaluate the viability of HT29 and HCT116 cells treated with Vemurafenib. Western blotting was performed to analyze the amount and/or the activity of mediators (LMWPTP, PTP1B, TGFβ, SMAD3), and the immunoprecipitation was performed to evaluate LMWPTP activity. This study brought up novel aspects of Vemurafenib action in colorectal cancer, which can decrease the activity of protein tyrosine phosphatases (LMWPTP and PTP1B) and the TGFβ pathway, making them important in the CRC aggressiveness. By downmodulating colorectal cancer hallmarks, Vemurafenib appears as an interesting candidate for CRC therapeutic protocols.