We have previously implicated calcium entry through stretch-activated cation channels in initiating the acute pulmonary vascular permeability increase in response to high peak inflation pressure (PIP) ventilation. However, the molecular identity of the channel is not known. We hypothesized that the transient receptor potential vanilloid-4 (TRPV4) channel may initiate this acute permeability increase because endothelial calcium entry through TRPV4 channels occurs in response to hypotonic mechanical stress, heat, and P-450 epoxygenase metabolites of arachidonic acid. Therefore, permeability was assessed by measuring the filtration coefficient (K_f) in isolated perfused lungs of C57BL/6 mice after 30-min ventilation periods of 9, 25, and 35 cmH₂O PIP at both 35°C and 40°C. Ventilation with 35 cmH₂O PIP increased K_f by 2.2-fold at 35°C and 3.3-fold at 40°C compared with baseline, but K_f increased significantly with time at 40°C with 9 cmH₂O PIP. Pretreatment with inhibitors of TRPV4 (ruthenium red), arachidonic acid production (methanandamide), or P-450 epoxygenases (miconazole) prevented the increases in K_f. In TRPV4^−/− knockout mice, the high PIP ventilation protocol did not increase K_f at either temperature. We have also found that lung distention caused Ca²⁺ entry in isolated mouse lungs, as measured by ratiometric fluorescence microscopy, which was absent in TRPV4^−/− and ruthenium red-treated lungs. Alveolar and perivascular edema was significantly reduced in TRPV4^−/− lungs. We conclude that rapid calcium entry through TRPV4 channels is a major determinant of the acute vascular permeability increase in lungs following high PIP ventilation.