Hypoxia and epithelial stretch that are commonly observed in patients with acute lung injury have been shown to promote the release of serotonin in vitro. However, whether serotonin contributes to the decrease of the alveolar epithelial fluid transport, which is a hallmark of lung injury, is unknown. Thus, we investigated the effect of serotonin on ion and fluid transport across the alveolar epithelium. Serotonin caused a dose-dependent inhibition of the amiloride-sensitive current across primary rat and human alveolar epithelial (ATII) cell monolayers, but did not affect Na+/K+ ATPase function. Furthermore, we found that the serotonin-induced inhibition of ion transport across the lung epithelium was receptor-independent because it was not prevented by the blockade of 5-HT2R, 5-HT3R or by pretreatment with an intracellular calcium-chelating agent, BAPTA-AM. In addition, the stimulation of 5-HT1R, 5-HT2R, 5-HT4R and 5-HT7R failed to reproduce the serotonin effect on amiloride-sensitive sodium transport. We ascertained that serotonin directly inhibited the function of rat ENaC as determined by heterologous expression of rat ENaC in Xenopus oocytes that do not express endogenous ENaC nor serotonin receptors. Exposure of mice to hypoxia for one hour induced a 30% increase of serotonin secretion into the distal airways of mice. Lastly, the intra-tracheal instillation of serotonin inhibited the amiloride-sensitive fraction of alveolar fluid clearance in mice. Taken together, these results indicate that serotonin inhibits the amiloride-sensitive fraction of the alveolar epithelial fluid transport via a direct interaction with the sodium channel ENaC and thus can be an endogenous inhibitor of this ion channel.