The microvascular endothelial cell monolayer localized at the critical interface between the blood and vessel wall has the vital functions of regulating tissue fluid balance and supplying the essential nutrients needed for the survival of the organism. The endothelial cell is an exquisite “sensor” that responds to diverse signals generated in the blood, subendothelium, and interacting cells. The endothelial cell is able to dynamically regulate its paracellular and transcellular pathways for transport of plasma proteins, solutes, and liquid. The semipermeable characteristic of the endothelium (which distinguishes it from the epithelium) is crucial for establishing the transendothelial protein gradient (the colloid osmotic gradient) required for tissue fluid homeostasis. Interendothelial junctions comprise a complex array of proteins in series with the extracellular matrix constituents and serve to limit the transport of albumin and other plasma proteins by the paracellular pathway. This pathway is highly regulated by the activation of specific extrinsic and intrinsic signaling pathways. Recent evidence has also highlighted the importance of the heretofore enigmatic transcellular pathway in mediating albumin transport via transcytosis. Caveolae, the vesicular carriers filled with receptor-bound and unbound free solutes, have been shown to shuttle between the vascular and extravascular spaces depositing their contents outside the cell. This review summarizes and analyzes the recent data from genetic, physiological, cellular, and morphological studies that have addressed the signaling mechanisms involved in the regulation of both the paracellular and transcellular transport pathways.