Mucin 1 (MUC1) is a large transmembrane glycoprotein that is aberrantly overexpressed in most adenocarcinomas and certain hematological malignancies. MUC1 is known to function as an oncogene with roles in both tumor formation and progression, making it a potential target for immunotherapy. B16-MUC1 cells with human full-length MUC1 are frequently used to study the antitumor activities of MUC1-based vaccines. However, we found that the growth of B16-MUC1 cells was significantly reduced in vitro. Therefore, in this study, we established two MUC1-positive clones, B16-MUC1 9-12 and B16-MUC1 9-23, and one empty vector control clone, B16-neo, to investigate the effects of MUC1 on the cancer-related characteristics of B16 cells in vitro and in vivo. Our results demonstrated that, compared with MUC1-negative cells, cells expressing MUC1 exhibited decreased cell proliferation, increased cell cycle arrest and reduced cell migratory and invasive capacities. We further investigated several MUC1-related molecules of the β-catenin pathway, and found that the expression of MUC1 decreased the translocation of β-catenin into the nucleus, reduced the activity of T cell factor (TCF) and blocked the expression of cyclin D1 and c-Myc. Moreover, when inoculated into BALB/c nude mice, cells expressing MUC1 developed smaller tumors compared with the control cells. These results demonstrate that MUC1 expression negatively affects the malignancy of B16 cells, and suggest that the regulatory mechanisms of MUC1 as an oncoprotein are more complex than previously appreciated.