Colonic fermentation products, SCFA, have various effects on colonic functions. Here, we found that physiological concentrations of SCFA immediately promote epithelial barrier function in the large intestine. Solutions of mixed and individual SCFA were applied to the caecal walls mounted on Ussing-type chambers. Transepithelial electrical resistance (TER) increased rapidly and reached a peak 35 % higher than that in the control specimen within 10 min post application of the SCFA mixture (80 acetate, 40 propionate, 20 butyrate (mmol/l)). The Lucifer yellow permeability, a paracellular transport marker, was dose-dependently reduced by the mixed SCFA, acetate and propionate solutions. Inhibition of monocarboxylate transporter-1 did not influence the increase in TER with acetate; however, lowering the pH (from 7.5 to 5.5) clearly enhanced the effect of acetate. Non-metabolizable, bromo and chloro derivatives of SCFA also increased TER. These results suggest that passive diffusion of SCFA is dominant and the metabolism of SCFA is not required for the promotive effect of SCFA on barrier function. We also observed that individual SCFA dose-dependently increased TER in T84 and Caco-2 cells, which indicates that SCFA directly stimulate epithelial cells. Depletion of membrane cholesterol and inhibitors of phosphatidylinositol-3 kinase and Gq protein attenuated the acetate-mediated promotive effect. Finally, we found that the mucosal application of the SCFA mixture dose-dependently suppressed [3H] mannitol transport from the caecal lumen to the mesenteric blood in the anaesthetized rats. We conclude that physiological concentrations of SCFA immediately enhance barrier function of the colonic epithelium through cholesterol-rich microdomain in the plasma membrane.