We examined how melatonin absorption was affected by pharmaceutical excipients using the Ussing chamber technique with mounted rat gastrointestinal (GI) segments. Melatonin absorption occurs throughout the GI tract, with the greatest absorption being in the rectum and ileum and the least in the stomach. Melatonin can be classified as a low permeability drug. P-glycoprotein (P-gp) does not affect melatonin absorption but transported rhodamine 123, a well-known P-gp substrate. The possibility of saturating P-gp by melatonin was excluded. Sodium cholate (0.5%) increased melatonin absorption, but decreased absorption at higher concentrations (1.0% and 5.0%). Sodium oleate (0.5% and 1.0%) consistently decreased melatonin absorption. Pharmaceutical excipients increased the absorption of Lucifer yellow (100 microg/mL), a paracellular probe but decreased the absorption of melatonin above the critical micelle concentration (cmc), suggesting that melatonin was transported mainly by transcellular pathway. Sodium cholate and sodium oleate, when above the cmc, resulted in micellar complexes as revealed by (1)H NMR spectra and particle size distribution. Histology tests showed mucosal damage of jejunum tissues in the presence of these excipients. The balance of tissue damage by the formation of micellar complexes could affect the melatonin absorption. This information on melatonin absorption behaviors and its modulation by pharmaceutical excipients can be used in further oral dosage formulations to affect circadian rhythm.