The MUC1 protein is expressed as a stable heterodimer from a single polypeptide, which was cleaved into two subunits in endoplasmic reticulum. It localizes at the cell membrane as an α/β-complex, tethered by the β-subunit transmembrane domain. Previous studies implicated that the three amino acids of the transmembrane domain adjacent to the cytoplasmic domain in MUC1 β-subunit are the residues Cys-Gln-Cys (CQC). Therein, site-directed mutagenesis of the CQC motif was performed and the cell lines were established. These cell lines include HCT116/MUC1, HCT116/MUC1(CQC→AQA), HCT116/MUC1(△CQCRRK), HCT116/MUC1C-ter(CQC→AQA), which can express wild type or mutant MUC1 on the cell surface，or its cytoplasmic domain. The effects of CQC→AQA mutation or CQCRRK deletion were investigated in vitro and in vivo. Compared with wild type MUC1, the mutants depressed soft agar colony formation and showed abrogated tumorigenicity in nude mice. These findings implicate that CQCRRK motif mediate the formation of MUC1 protein complex. As a result of this research, disruption of MUC1-C-terminal subunit-associated dimerization by mutation of CQC→AQA might represent a novel therapeutic approach for tumor.