The effect of prolactin (PRL) on ion transport across the rat colonic epithelium was investigated using the Ussing chamber technique. PRL induced a sustained decrease in basal short circuit current (Isc) by 5–35% depending on the colonic segment: a maximum response in the distal colon and less in the proximal colon. In the distal colon, PRL decreased Isc in a concentration dependent manner with an IC50value of 140 ng/ml. The PRL‐induced decrease in Isc was not affected in the presence of Na+ channel blocker amiloride, but showed higher response in the presence of Cl− channel blocker NPPB or glibenclamide added to the apical solution. In contrast, the PRL response was inhibited in the presence of Na+‐K+‐2Cl− transporter inhibitor bumetanide or K+ channel blocker BaCl2, but not other K+ channel blocker apamin, clotrimazole or 293B added serosally. Among K+ channel blockers, only 293B added apically partially diminished the PRL response. In addition, the carbachol or forskolin‐activated Cl− secretion remained unchanged in the presence of PRL. Thus in the rat distal colon, PRL stimulates K+ secretion through barium‐sensitive basolateral K+ channel and bumetanide‐sensitive pathway, with no effect on Na+ or Cl− transport. This work was supported by The Srinakharinwirot University Research Grant (325/2552).