A well-functional intestinal mucosal barrier can be compromised as a result of various diseases, chemotherapy, radiation, and chemical exposures including surfactants. Currently, there are no approved drugs targeting a dysfunctional intestinal barrier, which emphasizes a significant medical need. One candidate drug reported to regulate intestinal mucosal permeability is melatonin. However, it is still unclear if its effect is primarily receptor mediated or antioxidative, and if it is associated with enteric neural pathways. The aim of this rat intestinal perfusion study was to investigate the mechanisms of melatonin and nicotinic acetylcholine receptors on the increase in intestinal mucosal clearance of 51Cr-labeled ethylenediaminetetraacetate induced by 15 min luminal exposure to the anionic surfactant, sodium dodecyl sulfate. Our results show that melatonin abolished the surfactant-induced increase in intestinal permeability and that this effect was inhibited by luzindole, a melatonin receptor antagonist. In addition, mecamylamine, an antagonist of nicotinic acetylcholine receptors, reduced the surfactant-induced increase in mucosal permeability, using a signaling pathway not influenced by melatonin receptor activation. In conclusion, our results support melatonin as a potentially potent candidate for the oral treatment of a compromised intestinal mucosal barrier, and that its protective effect is primarily receptor-mediated.