Patients with metabolic syndrome have increased risk of uric acid nephrolithiasis due to lower urinary pH and impaired ammonium excretion. The pathophysiology underlying these urinary changes is unknown. We used two animal models and a cell culture model to study whether the alteration in renal acidification is associated with renal fat infiltration (steatosis). When compared to pair-fed lean control rats, Zucker diabetic fatty rats have higher renal triglyceride content, decreased urinary ammonium and pH, and lower levels of brush border membrane Na⁺/H⁺ exchanger NHE3, a major mediator of ammonium excretion. High-fat feeding in Sprague-Dawley rats results in transient lowering of urinary ammonium and pH with all parameters returning to normal when resumed on normal chow. This is consistent with absence of diet-induced renal steatosis in these animals. To examine the direct effect of fat accumulation, we incubated opossum kidney (OKP cells) with a mixture of long chain fatty acids and found accumulation of intracellular lipids with concomitant dose-dependent decrease in NHE3 activity, surface biotin-accessible NHE3 protein, and ammonium secretion. A lower dose of fatty acids that leads to intracellular lipid accumulation but does not change baseline NHE3, is sufficient to abolish the stimulation of NHE3 by insulin and to partially block the stimulation of NHE3 by glucocorticoid hormones; acid regulation of NHE3 in lipid-loaded OKP cells is not affected. These findings suggest that renal steatosis decreases ammonium secretion in the proximal tubule, in part by reducing NHE3 activity and by impairing the regulation of NHE3 by specific agonists.