Currently, 5-FU-based combination chemotherapy is the mainstay in the treatment of metastatic colorectal cancer (CRC) which benefits approximately 50% of patients. However, these tumours inevitably acquire chemoresistance resulting in treatment failure. Even though the molecular mechanisms driving acquired chemotherapeutic drug resistance in CRC is fundamental to the development of novel strategies for circumventing resistance, it is poorly understood. A preliminary quantitative proteomics analysis of parental and 5-FU resistant CRC cells identified the epigenetic modifier protein SMCHD1 to be upregulated upon acquiring chemotherapeutic resistance to 5-FU. To account for tumor heterogeneity, a panel of CRC cells were made resistant to 5-FU by long term exposure to increasing concentrations of the drug. Follow up Western blotting analysis confirmed the upregulation of SMCHD1 upon gaining chemoresistance. In order to further understand the role of SMCHD1, TALEN mediated knockout was performed in SW620 CRC cells that are resistant to 5-FU. Western blot and proteomic analysis highlighted that epithelial-to-mesenchymal (EMT) markers were regulated upon knockout of SMCHD1 implying that SMCHD1 regulates EMT. Co-immunoprecipitation assay followed by proteomic analysis was performed to identify the interacting partners of SMCHD1. These findings imply that treatment of cancer cells with chemotherapeutic drugs leads to EMT, increased migration and invasion.