Tissue barriers maintain homeostasis, protect underlying tissues, are remodeled during organogenesis and injury and limit aberrant proliferation and dissemination. In this context, endothelial and epithelial intercellular junctions are the primary targets of various cues. This cellular adaptation requires plasticity and dynamics of adhesion molecules and the associated cytoskeleton, as well as the adhesive-linked signaling platforms. It is therefore not surprising that the guidance molecules from the Semaphorin family arise as novel modifiers of epithelia and endothelia in development and diseases. This review will focus on the actions of Semaphorins, and their cognate receptors, Plexins and Neuropilins, on epithelial and endothelial barrier properties.