Mucin 1 (MUC1) is overexpressed in various human malignant tumors and its expression is correlated with a poor prognosis. MUC1 engages in signal transduction by interacting with receptors for growth and differentiation factors, which contributes to the growth and survival of cancer cells. However, the mechanism by which MUC1 promotes cancer cell invasion remains unclear. Microarray analysis revealed that expression of urokinase-type plasminogen activator (uPA) was elevated in MUC1-overexpressing cells. Furthermore, up- and down-modulation of MUC1 expression was clearly correlated with the change of uPA expression. An immunochemical study showed that the distribution of uPA coincided with that of MUC1 in various human cancer tissues. The MUC1 C-terminal domain (MUC1-CD) was associated with nuclear factor-κB (NF-κB) p65 in MUC1-expressing cells. Chromatin immunoprecipitation (ChIP) assays demonstrated that MUC1-CD existed with NF-κB p65 on the uPA promoter. Luciferase assays indicated that the uPA transcriptional activity was correlated with the level of MUC1 expression and that this MUC1-enhancing effect on the uPA transcription was abolished by introduction of mutations into the NF-κB binding sites on the uPA promoter. These results indicate that formation of the MUC1-CD and NF-κB p65 complex enhanced nuclear translocation of NF-κB p65 and subsequent occupancy of NF-κB binding region on the uPA promoter, leading to elevated transcription of uPA. We also demonstrated that uPA induced by MUC1 enhanced the matrix metalloproteinase (MMP)-2 and -9 activities, and consequently promoted cancer cell invasion. Thus, a MUC1 co-operating NF-κB signaling pathway plays a critical role in cancer cell invasion in MUC1-expressing cells.