Lung bacterial infections, including hospital-acquired pneumonia, remain a serious problem for public health. Endothelial cell (EC) exposure to heat-killed Staphylococcus aureus (HKSA) represents a clinical scenario of high titers of killed bacterial particles present in the host after antibiotic therapy, which triggers inflammatory cascades, cytokine storms, and EC dysfunction leading to acute lung injury (ALI). GPR68 is a member of the proton-sensing G protein-coupled receptor family. Acting as a pH sensor, GPR68 becomes activated upon pH reduction and contributes to pathologic cell responses by activating ER stress and unfolded protein response. This study investigated the role of GPR68 in HKSA-induced EC dysfunction and HKSA-induced ALI. HKSA robustly increased GPR68 mRNA levels in human pulmonary EC and directly stimulated GPR68 activity. A selective GPR68 small molecule inhibitor, OGM-8345, attenuated HKSA-induced EC permeability and protected cell junction integrity. OGM-8345 inhibited HKSA-induced activation of inflammatory genes TNF-α, IL-6, IL-8, IL-1β, and CXCL5 and decreased cytokine secretion by HKSA-challenged EC. Co-treatment with the GPR68 activator Ogerin or medium acidification to pH 6.5 augmented HKSA-induced EC dysfunction, which was rescued by OGM-8345. Intratracheal HKSA injection increased vascular leak and lung inflammation in mice which were monitored by lung Evans blue extravasation, increased cell and protein count in bronchoalveolar lavage, and mRNA expression of inflammatory genes. ALI and barrier dysfunction was attenuated by OGM-8345. We show for the first time the role of GPR68 in mediating HKSA-induced lung injury and the strong potential for OGM-8345 as a therapeutic treatment of bacterial pathogen-induced ALI associated with tissue acidification.