Bacterial induced sepsis is a common cause of pulmonary endothelial barrier dysfunction and can progress toward acute respiratory distress syndrome (ARDS). Elevations in intracellular cAMP tightly regulate pulmonary endothelial barrier integrity; however, cAMP signals are highly compartmentalized and it depends on which compartment the signal is generated - plasma membrane versus cytosolic - as to whether it is barrier protective or disruptive, respectively. The mammalian soluble adenylyl cyclase (AC) isoform 10 (AC10 or sAC) is uniquely stimulated by bicarbonate and is expressed in pulmonary microvascular endothelial cells (PMVECs). Elevated extracellular bicarbonate increases cAMP in PMVECs to disrupt the endothelial barrier and increase the filtration coefficient (K_f) in the isolated lung. Herein, we tested the hypothesis that sepsis-induced endothelial barrier disruption and increased permeability is dependent upon extracellular bicarbonate and activation of AC10. Our findings reveal that LPS-induced endothelial barrier disruption is dependent upon extracellular bicarbonate: LPS-induced barrier failure and increased permeability is exacerbated in elevated bicarbonate compared to low extracellular bicarbonate. The AC10 inhibitor, KH7, attenuated the bicarbonate-dependent LPS-induced barrier disruption. In the isolated lung, LPS failed to increase the K_f in the presence of minimal perfusate bicarbonate. When perfusate bicarbonate was increased to the physiological range (24 mM) this revealed the LPS-induced increase in the K_f, which was attenuated by KH7. Further, when PMVECs were treated with LPS for 6-hours there was a dose-dependent increase in AC10 expression. Thus, these findings reveal that LPS-induced pulmonary endothelial barrier failure requires bicarbonate activation of AC10.