Sepsis and endotoxemia induce defects in renal tubule function, but the mechanisms are poorly understood. Recently, we demonstrated that lipopolysaccharide (LPS) inhibits HCO₃⁻ absorption in the medullary thick ascending limb (MTAL) through activation of different Toll-like receptor 4 (TLR4) signaling pathways in the basolateral and apical membranes. Basolateral LPS inhibits HCO₃⁻ absorption through ERK-dependent inhibition of the apical Na⁺/H⁺ exchanger NHE3. Here, we examined the mechanisms of inhibition by lumen LPS. Adding LPS to the lumen decreased HCO₃⁻ absorption by 29% in rat and mouse MTALs perfused in vitro. Inhibitors of phosphoinositide 3-kinase (PI3K) or its effectors Akt and mammalian target of rapamycin (mTOR) eliminated inhibition of HCO₃⁻ absorption by lumen LPS but had no effect on inhibition by bath LPS. Exposure to LPS for 15 min induced increases in phosphorylation of Akt and mTOR in microdissected MTALs that were blocked by wortmannin, consistent with activation of Akt and mTOR downstream of PI3K. The effects of lumen LPS to activate Akt and inhibit HCO₃⁻ absorption were eliminated in MTALs from TLR4^−/− and MyD88^−/− mice but preserved in tubules lacking Trif or CD14. Inhibition of HCO₃⁻ absorption by lumen LPS was eliminated under conditions that inhibit basolateral Na⁺/H⁺ exchange and prevent inhibition of HCO₃⁻ absorption mediated through NHE1. Lumen LPS decreased basolateral Na⁺/H⁺ exchange activity through PI3K. We conclude that lumen LPS inhibits HCO₃⁻ absorption in the MTAL through TLR4/MyD88-dependent activation of a PI3K-Akt-mTOR pathway coupled to inhibition of NHE1. Molecular components of the TLR4-PI3K-mTOR pathway represent potential therapeutic targets for sepsis-induced renal tubule dysfunction.