Abstract Fermentation of dietary fibres by colonic microbes leads to the production of short chain fatty acids (mainly propionate, butyrate and acetate), which are utilized by the colonic mucosa. Previous studies showed positive effects of butyrate on parameters of oxidative stress, inflammation and apoptosis. Recent studies in rats, however, showed that butyrate increased visceral sensitivity. The aim of this study was to determine the effects of physiologically relevant concentrations of butyrate on visceral perception in healthy human subjects. Eleven healthy volunteers participated in this randomized double-blind, placebo controlled cross-over study. The study consisted of three periods of 1 week each, in which the volunteers daily self-administered rectal enemas containing 100, 50 mmol L−1 butyrate, or placebo (saline) prior to sleeping. A rectal barostat measurement was performed at the start and the end of each test period for the measurement of pain, urge and discomfort. Butyrate treatment resulted in a dose-dependent reduction of pain, urge and discomfort throughout the entire pressure range of the protocol. At a pressure of 4 mmHg, 50 and 100 mmol L−1 butyrate concentrations resulted in a 23.9% and 42.1% reduction of pain scores, respectively, and the discomfort scores decreased by 44.2% and 69.0% respectively. At a pressure of 67 mmHg, 50 and 100 mmol L−1 of butyrate decreased the pain scores by 23.8% and 42%, respectively, and discomfort scores 1.9% and 5.2% respectively. Colonic administration of butyrate, at physiologically relevant concentrations, dose-dependently decreases visceral sensitivity in healthy volunteers.