Diarrhoea complicates enteral feeding in up to 25% of patients. In-vivo perfusion studies in healthy subjects have shown secretion of salt and water in the ascending colon in response to enteral feeding. This study investigated the effect of short-chain fatty acids (SCFA) on this secretory response. Six healthy volunteers underwent segmental in-vivo colonic perfusion. First, baseline fasting colonic water and electrolyte movement was established, then a standard polymeric enteral diet was infused into the stomach while the colon was perfused with either a control electrolyte solution or a test solution containing SCFA. The electrolyte concentrations and osmolality of the two perfusates were identical. In the fasting state water was absorbed throughout the colon. During the control infusion there was significant (p < 0.05) secretion of water in the ascending colon (median rate 1.0 mL per min [95% CI 2.8 mL per min secretion to 0.8 mL per min absorption]). During the SCFA infusion the secretion was significantly reversed (p < 0.05) and there was net absorption (1.6 [0.8-3.7] mL per min). In the distal colon water absorption was significantly greater during the control infusion than during fasting (3.7 [2.5-4.6] vs 1.3 [0.3-2.2] mL per min); during the test infusion this absorption persisted (2.8 [1.3-3.6] mL per min). Movement of sodium, chloride, and potassium ions was similar to that of water in all stages of the study. Bicarbonate movement did not significantly change at any stage. Infusion of SCFA directly into the caecum reverses the fluid secretion seen in the ascending colon during enteral feeding. This finding could have implications for the management of diarrhoea related to enteral feeding.