Dendritic cells (DC) are potent professional antigen-presenting cells that drive primary immune responses to infections or other agonists perceived as 'dangerous'. Muc1 is the only cell surface mucin or MUC gene product that is expressed in DC. Unlike other members of this glycoprotein family, Muc1 possesses a unique cytosolic region capable of signal transduction and attenuating toll-like receptor (TLR) activation. The expression and function of Muc1 has been intensively investigated on epithelial and tumor cells, but relatively little is known about its function on DC. We hypothesized that Muc1 would influence in vitro generation and primary DC activation in response to the TLR4 and TLR5 ligands lipopolysaccharide and flagellin. Compared with Muc1(+/+) DC, we found that Muc1(-/-) DC were constitutively activated, as determined by higher expression of co-stimulatory molecules (CD40, CD80 and CD86), greater secretion of immunoregulatory cytokines (TNF-alpha and VEGF), and better stimulation of allogeneic naïve CD4+ T cell proliferation. After activation by either LPS or flagellin and co-culture with allogeneic CD4+ T cells, Muc1(-/-) DC also induced greater secretion of TNF-alpha and IFN-gamma compared to similarly activated Muc1(+/+) DC. Taken together, our results indicate that deletion of Muc1 promotes a heightened functional response of DC in response to TLR4 and TLR5 signaling pathways, and suggests a previously under-appreciated role for Muc1 in regulating innate immune responses of DC.