High-altitude pulmonary edema (HAPE) is an increasing problem with high morbidity and mortality due to its rapid onset and progression. Perfluorocarbon (PFC) is reported to have promising effects on acute lung injury (ALI) by offering oxygenation and anti-inflammatory effects. However, PFC's effect on HAPE is still unknown. In our study, we investigated the effect of PFC, first in a HAPE canine model and then in a lung-on-chip model. We exposed beagle dogs to a simulated altitude of 6000 m for 48 h to establish the HAPE model and treated them with vaporized PFC. Next, we created a lung-on-chip model that allowed lung injury to be recapitulated in vitro at the organ level. Then, the latter model was exposed to hypoxia (3% oxygen; 24 h) and co-cultured with 10% PFC. As expected, hypoxia caused an increase in lung water content, lung injury scores, and inflammatory cytokine release, as well as a decrease in blood–gas barrier–related proteins. Treatment with PFC could inhibit the inflammatory response and alleviate blood–gas barrier disruption in our canine and lung-on-chip models. In addition, we found that PFC blocked activation of the hypoxia-inducible factor-1α (HIF-1α)/heme oxygenase-1 (HO-1) signaling pathway. PFC offers great potential in treating hypoxia-induced lung injury, mainly by suppressing dysfunction of the blood–gas barrier via the HIF-1α/HO-1 pathway.