Objective ·To identify the functional motifs of mucin 1 (MUC1) involved in regulating tumor cell proliferation, migration, and stemness maintenance. Methods ·Mutational characteristics of the MUC1 gene across different cancers were identified from The Cancer Genome Atlas (TCGA) database. Various MUC1 mutation sites were analyzed and localized, followed by ranking based on mutation frequency. Western blotting was used to screen high-frequency MUC1 mutants with stable protein expression. BT549 cell line with MUC1 knocked out and MCF-10A cell line were used to stably overexpress MUC1 wild-type (MUC1-WT) and mutants by using lentiviral technology. Immunofluorescence was used to detect the cellular localization of MUC1 mutants. Using MUC1-WT as a positive control and MUC1-AQA, a loss-of-function mutant, as a negative control, the biological functions of different MUC1 mutant cells were analyzed: cell proliferation ability was assessed by cell counting kit-8 (CCK-8) assay and colony formation assay; cell migration ability was evaluated by wound-healing and Transwell assays; cell stemness was examined by sphere formation assay. Structural localization of MUC1 mutants was analyzed by using PyMOL software, and molecular docking analysis was performed by using a protein docking software (ZDOCK Server). ·Results A total of 102 mutations located in the MUC1 coding region were identified in the TCGA database, among which five missense mutations (P418S, S251R, V359I, N271S, and N465H) exhibited higher frequencies and were located in the non-variable number of tandem repeats (non-VNTR) region. Further examination revealed that the MUC1-S251R, N271S, and V359I mutants could be stably expressed. The cellular localization assay indicated that these three mutants predominantly localized in the cytoplasm, but were also presented in the nucleus. The nuclear-to-cytoplasmic ratio showed minimal differences between MUC1-WT and the mutants. Analysis of the tumorigenic biological functions of the cells expressing different MUC1 mutants revealed that: ① High expression of MUC1-WT significantly enhanced the proliferation ability of both BT549 and MCF-10A cells; the proliferation of MUC1-AQA, S251R, and N271S mutant cells was decreased compared to MUC1-WT cells, while MUC1-V359I mutant cells exhibited a similar proliferative profile to MUC1-WT cells. ② The migration ability of MUC1-WT high-expressing cells was significantly enhanced, whereas MUC1-AQA cells demonstrated attenuated migration. In the BT549 cells, the migration ability of MUC1-S251R and V359I cells was similar to that of MUC1-WT cells, whereas MUC1-N271S cells showed reduced migration. In the MCF-10A cells, the migration ability of MUC1-N271S and MUC1-V359I cells was similar to that of MUC1-WT cells, whereas MUC1-S251R cells exhibited significantly decreased migration. ③ Stemness was enhanced in both cell types with high MUC1-WT expression, while MUC1-AQA cells lost stemness; the cells with MUC1-N271S, V359I and MUC1-WT showed comparable maintenance of stemness, whereas MUC1-S251R cells demonstrated compromised stemness. PyMOL software analysis unveiled that MUC1-N271S and V359I were located in or around the sperm protein-enterokinase-agarin (SEA) region, specifically in the loop region and the β-sheet, respectively. The molecular docking analysis revealed that the stability of the complex formed by MUC1-WT or V359I with the extracellular domain of epidermal growth factor receptor (EGFR) surpassed that of MUC1-N271S or S251R, indicating a stability hierarchy of V359I>WT>N271S>S251R. ·Conclusion MUC1 mutants exhibit diverse impacts on the biological functions of tumor cells, with their effects on proliferation correlating with the EGFR signaling pathway. MUC1-V359I is similar to MUC1-WT, indicating a negligible effect on tumor cell proliferation, migration, and stemness maintenance. Conversely, MUC1-S251 and N271 sites may be involved in the regulation of signaling pathways governing cell proliferation and migration and the MUC1-S251 site plays a critical role in maintaining cell stemness.