Five year survival rates for patients with metastatic colorectal cancer remains below 15%, necessitating an urgency to develop novel therapeutic approaches for this disease. Differentiation therapy has been successful in the treatment of acute promyelocytic leukemia, however whether this therapy is efficacious in colon cancer is unknown. Our laboratory has previously demonstrated that Histone deacetylase inhibitors (HDACi) can induce differentiation of colorectal cancer cells. In addition, recent findings demonstrate that inhibition of the MAPK pathway can also induces differentiation of colon cancer cells. This study aims to determine whether combinatorial treatment with HDACi and MAPKi can additively induce differentiation of colon cancer cells and whether this promotes the induction of a terminal differentiation program as assessed by the induction of apoptosis. Combination treatment of the colon cancer cell lines HT29 and T84 with HDACi and MAPKi induced significantly greater expression of the differentiation markers Cadherin17, Keratin20 and Alkaline phosphatase, compared to either agent alone. This combination also increased expression of the CDX2 transcription factor, a known regulator of colon cell differentiation. This drug combination also significantly reduced the number of proliferating cells and significantly increased apoptosis. Furthermore, the effect on apoptosis was consistently observed in response to other combinations of HDACi (SAHA, Panobinostat and Entinostat) and MAPK pathway inhibitors (Cetuximab and Regorafenib). In contrast, the poorly differentiated RKO cell line was less responsive to both, differentiation-inducing and apoptotic effects of the combination. Changes in cell proliferation and apoptosis induced by this combination treatment were associated with changes in expression of known regulators of cell proliferation (p57, c-MYC) and apoptosis (BMF, PUMA). Whether these genes directly mediates cell proliferation and apoptosis remains to be determined. This study identifies a novel drug combination that may benefit the treatment of colon cancer through induction of differentiation, growth inhibition and apoptosis of colon cancer cells.