We previously reported by using intravital imaging technique that lipopolysaccharide (LPS) slowed proximal tubular flow rate in an early phase of endotoxemia. Hereby, we hypothesized that LPS disrupts tight junction in proximal tubular cells and induce leakage of filtrate through a Toll-like receptor 4 (TLR4)-dependent mechanism. LPS at 5 mg/kg did not change glomerular filtration rate (GFR), and significantly reduced the washout rate of tubular fluid from the proximal tubules and urine output in the early phase, reflecting slowing down of tubular flow rate in the proximal tubules during oliguria. LPS at 15 mg/kg reduced both GFR and urine output. LPS (5 mg/kg) induced paracellular leakage of FITC-inulin and reduced tight junction mRNA expression (occludin and cldn2). LPS also increased water content, interstitial hydrostatic pressure and Na⁺/K⁺ ratio in the kidney, indicating the accumulation of extracellular fluid in the interstitium. The mice lacking TLR4 in proximal tubules showed markedly blunted aforementioned responses and an increased sensitivity to the fluid resuscitation. Our results suggest that LPS disrupted tight junction of proximal tubular cells via a TLR4-dependent mechanism, resulting in paracellular leakage of filtrate to interstitium, which blunted fluid sensitivity, during endotoxemia in mice.