Colonic fermentation of carbohydrate has been shown to influence gastric and intestinal motility. Our aim was to investigate the effects of colonic infusion of lactose and short-chain fatty acids (SCFAs) on lower esophageal sphincter (LES) function in humans. LES pressure (LESP), transient relaxations of LES (TLESRs), and esophageal pH were monitored over 6 h on 4 different days in 7 healthy volunteers. After 1 h of baseline recording, the effects of different colonic infusions (270 ml of isotonic or hypertonic saline, 30 g lactose, or 135 mmol SCFAs) were tested in fasting conditions and after a standard meal. Peptide YY (PYY) and oxyntomodulin (OLI) were also measured in plasma. Both lactose and SCFA infusions increased the number of TLESRs as well as the proportion of TLESRs associated with acid reflux episodes, but saline solutions did not. The postprandial fall of LESP was enhanced by previous SCFA infusion. Plasma PYY and OLI increased similarly after all colonic infusions. Colonic fermentation of lactose markedly affected LES function, and this effect was reproduced by SCFA infusion. Whether the mechanisms of this feedback phenomenon are of hormonal nature, neural nature, or both remains to be determined.