Lumen establishment and maintenance are fundamental for tubular organs physiological functions. Most of the studies investigating the mechanisms regulating this process have been carried out in cell cultures or in smaller organisms, whereas little has been done in mammalian model systems in vivo. Here we used the salivary glands of live mice to examine the role of the small GTPase Cdc42 in the regulation of the homeostasis of the intercellular canaliculi, a specialized apical domain of the acinar cells, where protein and fluid secretion occur. Depletion of Cdc42 in adult mice induced a significant expansion of the apical canaliculi, whereas depletion at late embryonic stages resulted in a complete inhibition of their postnatal formation. In addition, intravital subcellular microscopy revealed that reduced levels of Cdc42 affected membrane trafficking from and toward the plasma membrane, highlighting a novel role for Cdc42 in membrane remodeling through the negative regulation of selected endocytic pathways.