The biological responses that control the development of Transfusion-Related Acute Lung Injury (TRALI), a serious post-transfusion respiratory syndrome, still need to be clarified. Since extracellular nucleotides and their P2 receptors participate in inflammatory processes as well as in cellular responses to stress, we investigated the role of the ATP-gated P2X1 cation channel in antibody-mediated TRALI. The effects of NF449, a selective P2X1 receptor (P2RX1) antagonist, were analyzed in a mouse two-hit model of TRALI. Mice were primed with lipopolysaccharide (LPS) and 24 h later challenged by administrating an anti-MHC I antibody. The selective P2RX1 antagonist NF449 was administrated before the administration of LPS and/or the anti-MHC I antibody. When given before antibody administration, NF449 improved survival while maximal protection was achieved when NF449 was also administrated before the sensitization step. Under this later condition, protein contents in bronchoalveolar lavages were dramatically reduced. Cell depletion experiments indicated that monocytes/macrophages, but not neutrophils, contribute to this effect. In addition, the reduced lung periarteriolar interstitial edemas in NF449-treated mice suggested that P2RX1 from arteriolar smooth muscle cells could represent a target of NF449. Accordingly, inhibition of TRPC6, another cation channel expressed by smooth muscle cells, also reduced TRALI-associated pulmonary interstitial and alveolar edemas. These data strongly suggest that cation channels like P2RX1 or TRPC6 participate to TRALI pathological responses.