The medical treatment of colorectal cancer (CRC) has evolved greatly in the last years, involving combined chemotherapy protocols and, more recently, new biologic agents. Nevertheless, prognosis remains adverse for patients with metastatic disease and a significant portion of early-stage patients develop recurrence after chemotherapy. Clinical trials are now directed to evaluate new drug combinations and treatment schedules in order to overcome the mechanisms of chemoresistance. By the use of two patient-derived colon cancer cell lines, CC09 (BRAFV600E) and R511 (wt BRAF), and the established colon cancer cell line HT29 (BRAFV600E ; PIK3CAP449T), we found that the tyrosine kinase receptor HER3 is strongly involved in the mechanisms of resistance to 5FluoroUracil (5-FU) and Oxaliplatin drugs. By the use of a monoclonal antibody targeting HER3, named U3-1287, we found down-regulation of HER3 phosphorylation, HER3 internalization and degradation in all cell lines. Functionally, U3-1287 inhibits tumor cell proliferation inducing growth arrest in the G1 phase of the cell cycle, and reduces tumor mass in a CC09-derived xenograft model. Even though U3-1287 administration is higly efficient in abrogating the HER3-dependent activation of PI3K pathway in colon cancer cells, we also found that it induces a compensatory mechanism, involving the increase of HER2 receptor expression that in turn activates MAPK pathway. To overcome U3-1287-induced activation of MAPK, we used a combination therapy with U3-1287 antibody and the MEK-inhibitor Trametinib. We show that Trametinib alone induces the phosphorylation of HER3 receptor that in turn activates PI3K pathway; the combination therapy results in the complete abrogation of both PI3K and MAPK pathways, and in a significant reduction of cell survival in vitro in all three cancers cell lines. These data identify a new combination strategy that, independently of the genetic background of the cells, may overcome the mechanisms of resistance to chemotherapy in HER3-overexpressing colon cancers.