In ARDS, both reactive oxygen species (ROS) and increased intracellular calcium ([Ca²⁺]_i) are thought to play important roles in promoting endothelial paracellular permeability, but the mechanisms linking ROS and [Ca²⁺]_i in microvascular endothelial cells are not known. In this study, we assessed the effect of hydrogen peroxide (H₂O₂) on intracellular calcium ([Ca²⁺]_i) in mouse and human lung microvascular endothelial cells (MLMVEC and HLMVEC, respectively). We found that in both MLMVECs and HLMVECs, exogenously applied H₂O₂ increased [Ca²⁺]_i through Ca²⁺ influx and that pharmacologic inhibition of the calcium channel TRPV4 attenuated the H2O2-induced Ca²⁺ influx. Additionally, knockdown of TRPV4 in HLMVEC also attenuated calcium influx following H₂O₂ challenge. Administration of H₂O₂ or TRPV4 agonists decreased transmembrane electrical resistance (TER), suggesting increased barrier permeability. To explore the regulatory mechanisms underlying TRPV4 activation by ROS, we examined H₂O₂-induced Ca²⁺ influx in MLMVECs and HLMVECs with either genetic deletion, silencing or pharmacologic inhibition of Fyn, a Src family kinase. In both MLMVECs derived from mice deficient for Fyn and HLMVECs treated with either siRNA targeted to Fyn or the Src family kinase inhibitor, SU-6656, for 24 or 48 h, the H2O2-induced Ca²⁺ influx was attenuated. Treatment with SU-6656 decreased the levels of phosphorylated, but not total, TRPV4 protein and had no effect on TRPV4 response to the external agonist, GSK1016790A. In conclusion, our data suggest that application of exogenous H₂O₂ increases [Ca²⁺]_i and decreases TER in microvascular endothelial cells via activation of TRPV4 through a mechanism that requires the Src kinase Fyn.