CD36 belongs to class B of scavenger receptors and is a fatty acid translocase. Previous studies have suggested that the receptor may be involved in regulation of responses to endotoxin and inflammation of the lungs. We have tested a group of helical peptides that interact with and modulate the activities of CD36 for their activities against endothelial barrier dysfunction and acute lung injuries in mice. Peptides that inhibit CD36 activities including L37pA, ELR-B and ELR-BP reversed or attenuated the barrier disruptive activities induced by LPS, truncated phospholipids and histone H3 as measured by TER (trans-endothelial electric resistance). In consistence with these, the peptides also enhanced the barrier protective activities of OxPAPC. The same set of peptides inhibited LPS-induced expression of inflammatory markers including ICAM-1 and VCAM-1 through down-regulation of NFκB and State3 mediated signaling pathways in cultured endothelial cells. The peptides were delivered intravenously to mice treated with LPS to induce acute lung injury. It has been observed that among peptides that antagonize the activities of CD36, L37pA, ELR-B, ELR-P and ELR-BP have demonstrated inhibitory effects on parameters of acute lung injury induced by LPS characterized by reduced extravasation of inflammatory cells from circulation to bronchoalveolar lavage (BAL) fluid and a decrease in protein concentration in the BAL in peptide-treated mice. These results demonstrate for the first time the utility of CD36 antagonistic amphipathic helical peptides L37pA, ELR-B, ELR-P and ELR-BP as a novel strategy to ameliorate acute lung injury