Sepsis and septic acute lung injury are among the leading causes for morbidity and mortality of critical illness. Extracellular adenosine is a signaling molecule implicated in the cellular adaptation to hypoxia, ischemia, or inflammation. Therefore, we pursued the role of the A2B adenosine receptor (AR) as potential therapeutic target in endotoxin-induced acute lung injury. We gained initial insight from in vitro studies of cultured endothelia or epithelia exposed to inflammatory mediators showing time-dependent induction of the A2BAR (up to 12.9 ± 3.4-fold, p < 0.05). Similarly, murine studies of endotoxin-induced lung injury identified an almost 4.6-fold induction of A2BAR transcript and corresponding protein induction with LPS exposure. Studies utilizing A2BAR promoter constructs and RNA protection assays indicated that A2BAR induction involved mRNA stability. Functional studies of LPS-induced lung injury revealed that pharmacological inhibition or genetic deletion of the A2BAR was associated with dramatic increases in lung inflammation and histologic tissue injury. Studies of A2BAR bone marrow chimeric mice suggested pulmonary A2BAR signaling in lung protection. Finally, studies with a specific A2BAR agonist (BAY 60-6583) demonstrated attenuation of lung inflammation and pulmonary edema in wild-type but not in gene-targeted mice for the A2BAR. These studies suggest the A2BAR as potential therapeutic target in the treatment of endotoxin-induced forms of acute lung injury.