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 post-natal formation. In addition, intravital subcellular microscopy revealed that reduced levels of Cdc42 affected membrane trafficking from and towards the plasma membrane, highlighting a novel role for Cdc42 in membrane remodeling through the negative regulation of selected endocytic pathways. Movie S1 Movie S1 Movie S1. Volume rendering derived from a Z-stack phalloidin-stained acini. Cre-positive cells (green), Cre-negative cells (red), IC (yellow). Movie S2 Movie S2 Movie S2. Left: Time-lapse of 3 kDa Cascade Blue-dextran diffusion into the IC of adeno-Cre transfected Cdc42 visualized ISMic (5 sec/frame). Right: Maximal projection of a Z-stack acquired 180 sec after the injection of the dextran. Cre-positive cells (green), Cre-negative cells (red), Dextran (White). Movie S3 Movie S3 Movie S3. ISMic of representative Cre-positive cells where mGFP-labeled vesicles are tracked as described in Material and Methods. Arrows highlight the trajectories of selected vesicles. Basal membrane (BM), apical Plasma Membrane APM (II), lateral membrane (LM). Frame rate 2s per frame. Movie S4 Movie S4 Movie S4. ISMic of regulated exocytosis of secretory granules in mT/mG (Cdc42+) and Cdc42 (Cdc42 -/-) mice transfected with Adeno-Cre. Low magnification (Upper movies) and insets (lower movies) showing the integration of the secretory granules into the APM. Movie S5 Movie S5 Movie S5. Volume rendering derived from a Z-stack of phalloidin-stained acini in SMGs excised at postnatal day 5 (P5, left) and week 28 (Adult, right) from mTmG mouse (Cdc42+) and Cdc42 /AQP5-Cre mice (Cdc42-). Cre-positive cells (green), Cre-negative cells (red), IC (yellow). Movie S6 Movie S6 Movie S6. (related to Fig. S4) -Retrograde delivery of 3 kDa Cascade Blue-Dextran into the IC of Cdc42 /AQP5-Cre mice visualized by ISMic (2 sec/frame).