CyA was prepared into CyA Eudragit S100 nanoparticles (CyA-S100 NP) and the mechanisms of CyA-S100 NP improving the CyA absorption in gastrointestinal tract (GI) were studied systematically in rats. In the GI distribution study, the emptying rates of CyA-S100 NP in duodenum, jejunum, ileum and colon were all lower than these of Neoral, while in stomach, it was larger than that of Neoral. In in situ recirculating intestine perfusion experiment, the largest absorption in CyA-S100 NP group occurred in ileum while that in Neoral group arised in duodenum. The sequence of (AUC0-240 min)/A for CyA-S100 NP and Neoral group was ileum > duodenum > jejunum > colon and duodenum > jejunum > ileum > colon, respectively. CyA in nanoparticles degradated by luminal contents and subcellular fractions were more slowly than these in Neoral, suggesting the significant protect effect of nanoparticles. Mucoadhesion study in small intestine showed that among all the parts of intestine, CyA-S100 NP exhibited larger mucoadhesive characteristics than Neoral microemulsion. The sequence of mucoadhesion for CyA-S100 NP group was duodenum > ileum > jejunum and colon, while that for Neoral group was duodenum > ileum, jejunum and colon, suggesting different site-specific behaves. These results illustrated that nanoparticles increased the absorption of CyA could be attributed to fast stomach empting rate, absorption site specific, small degradation rate by luminal contents, high bioadhension of nanoparticles to intestine mucosa and the use of P-Glycoprotein inhibitor if there is any. This investigation is helpful for the dosage form design for other peptide or protein drugs.