Volume and composition of saliva are crucial for oral and systemic health. How substances, particularly macromolecules, are transported across the salivary gland epithelium has not been established in detail. Tricellulin is a component of tricellular tight junctions that form a central tube to serve as an important route for macromolecule transport. Whether tricellulin is expressed in the submandibular gland (SMG) and involved in salivation has been unknown. Here, by using Western blotting and immunofluorescence, tricellulin was found to be characteristically localized at tricellular contacts of human, rat, and mouse SMGs. Knockdown of tricellulin significantly increased, whereas overexpression of tricellulin decreased, paracellular permeability for 40-kDa but not for 4-kDa fluorescein isothiocyanate-dextran, while transepithelial electrical resistance was unaffected. Conversely, claudin-4 knockdown and overexpression affected transepithelial electrical resistance but not 40-kDa fluorescein isothiocyanate-dextran transport, suggesting that tricellulin regulated transport of macromolecules but not ions, which were mainly regulated by bicellular tight junctions (bTJs). Moreover, tricellulin was dynamically redistributed from tri- to bicellular membranes in cholinergically stimulated SMG tissues and cells. Immunoglobulin-like domain-containing receptor 1 (ILDR1) recruits tricellulin to tricellular contacts. The proportion of macromolecules in the saliva was increased, whereas the amount of stimulated saliva was unchanged in mice, which displayed abnormal tricellulin distribution in SMGs. Furthermore, tricellulin interacted with bTJ proteins, such as occludin, claudin-1, claudin-3, claudin-4, and ZO-1, in rat SMG epithelial polarized cell line SMG-C6. Knockdown of tricellulin decreased occludin levels. Thus, we revealed a specific expression pattern of tricellulin in SMG epithelium. Tricellulin not only functioned as a barrier for macromolecules but also modulated the connection of bTJs to the tight junction complex. Alterations in tricellulin expression and distribution could thereby change salivary composition. Our study provided novel insights on salivary gland tight junction organization and function.