Pulmonary vascular endothelial cells express a variety of ion channels that mediate Ca²⁺ influx in response to diverse environmental stimuli. However, it is not clear whether Ca²⁺ influx from discrete ion channels is functionally coupled to specific outcomes. Thus we conducted a systematic study in mouse lung to address whether the α_1G T-type Ca²⁺ channel and the transient receptor potential channel TRPV4 have discrete functional roles in pulmonary capillary endothelium. We used real-time fluorescence imaging for endothelial cytosolic Ca²⁺, immunohistochemistry to probe for surface expression of P-selectin, and the filtration coefficient to specifically measure lung endothelial permeability. We demonstrate that membrane depolarization via exposure of pulmonary vascular endothelium to a high-K⁺ perfusate induces Ca²⁺ entry into alveolar septal endothelial cells and exclusively leads to the surface expression of P-selectin. In contrast, Ca²⁺ entry in septal endothelium evoked by the selective TRPV4 activator 4α-phorbol-12,13-didecanoate (4α-PDD) specifically increases lung endothelial permeability without effect on P-selectin expression. Pharmacological blockade or knockout of α_1G abolishes depolarization-induced Ca²⁺ entry and surface expression of P-selectin but does not prevent 4α-PDD-activated Ca²⁺ entry and the resultant increase in permeability. Conversely, blockade or knockout of TRPV4 specifically abolishes 4α-PDD-activated Ca²⁺ entry and the increase in permeability, while not impacting depolarization-induced Ca²⁺ entry and surface expression of P-selectin. We conclude that in alveolar septal capillaries Ca²⁺ entry through α_1G and TRPV4 channels differentially and specifically regulates the transition of endothelial procoagulant phenotype and barrier integrity, respectively.