5-hydroxytryptamine (5-HT)-evoked intestinal secretion can be divided into neural and non-neural pathway. Recently, 5-HT(3) receptor in neural pathway received much attention as a possible target in bowel diseases. The present study aims to investigate the effects of 5-HT(3) receptor in different enteric neural plexus (myenteric plexus and submucosal plexus) on rat colonic ion transport by using rat intact colon and mucosa/submucosa preparations. Ussing chamber and real-time PCR techniques were performed in our present study. Surprisingly, we found that in mucosa/submucosa preparations, 5-HT-induced DeltaI(SC) (change in short-circuit current) was not inhibited, but further increased by pretreatment with 5-HT(3) receptor antagonists, MDL72222 and Tropanyl-3, 5-dimethylbenzoate. And this response was significantly attenuated in the presence of tetrodotoxin (TTX). Conversely, in rat intact colon, 5-HT(3) receptor antagonists significantly inhibited 5-HT-induced DeltaI(SC). The results from real-time PCR proved the existence of 5-HT(3) receptor in muscularis externa and submucosa. Taken together, 5-HT(3) receptors possess a role of dual regulation on electrolyte secretion in rat distal colon, the neural stimulatory effect of 5-HT(3) receptor in myenteric plexus and the inhibitory effect of 5-HT(3) receptor in submucosal plexus.