Sepsis is the leading cause of acute kidney injury in critically ill patients. Tumor necrosis factor-α (TNF-α) is implicated in the pathogenesis of septic kidney injury; however, the sites and mechanisms of renal TNF-α production during sepsis remain to be defined. In this study, we show that TNF-α expression is increased in medullary thick ascending limbs (MTAL) of mice with sepsis induced by cecal ligation and puncture. Treatment with LPS for 3 h in vitro also increased MTAL TNF-α production. Sepsis and LPS increased MTAL TNF-α expression through activation of an MyD88-IRAK-1-ERK signaling pathway. Pretreatment with monophosphoryl lipid A (MPLA), a nontoxic immunomodulator that protects against bacterial infection, eliminated the sepsis- and LPS-induced increases in MTAL TNF-α production. The suppressive effect of MPLA on TNF-α was mediated through activation of a phosphatidylinositol 3-kinase (PI3K)-dependent pathway that inhibits MyD88-dependent ERK activation. This likely involves MPLA-PI3K-mediated induction of Tollip, which negatively regulates the MyD88-ERK pathway by inhibiting activation of IRAK-1. These regulatory mechanisms are similar to those shown previously to mediate the effect of MPLA to prevent sepsis-induced inhibition of MTAL HCO3- absorption. These results identify the MTAL as a site of local TNF-α production in the kidney during sepsis and identify molecular mechanisms that can be targeted to attenuate renal TNF-α expression. The ability of MPLA pretreatment to suppress MyD88-dependent ERK signaling in the MTAL during sepsis has the dual beneficial effects of protecting tubule transport functions and attenuating harmful proinflammatory responses.