Background: The aim of this study is to investigate the feasibility of three-layered particles as a drug delivery system to the lower part of small intestine. Methods: The particle surface and basement layers were made of enteric polymer, Eudragit® S100, and water-insoluble polymer, ethylcellulose. Prednisolone (PSL), as a model drug, was sealed with the surface and basement layers. After the administration of the test preparations to the duodenum of rats, blood samples were collected and plasma PSL levels were measured by high-performance liquid chromatography. The retention and transit characteristics of the three-layered particles in rat small intestine were studied by direct observation after abdominal incision up to 8 hours. Results: Three-layered PSL particles showed C(max) of 0.32 ± 0.07 μg/mL and T(max) at 6 hours, whereas the mean C(max) and T(max) of PSL powder, as a reference preparation, were 0.42 ± 0.03 μg/mL and 1 hour, respectively. With the direct observations, after administration of particles, about 77.5% of them were detected in duodenum at 1 hour, 45% in distal jejunum at 3 hours, and 50% in proximal ileum at 4 hours. Then, they were gradually transferred to the lower part of the small intestine at 5-8 hours time intervals. In comparison with PSL powder, three-layered particles delayed the intestinal transit and released PSL during their passage through the small intestine. Conclusion: These results suggested that three-layered particles adhered to the gastrointestinal mucosa and sustained the release of drug, resulting in drug delivery to the lower part of gastrointestinal tract.