Background: We previously reported that oral formulations containing indomethacin nanoparticles (IND-NPs) showed high bioavailability, and, consequently, improved therapeutic effects and reduced injury to the small intestine¹⁾. However, the pathway for the transintestinal penetration of nanoparticles remained unclear. Thus, in this study, we investigated whether endocytosis was related to the penetration of IND-NPs (72.1 nm) using a transcell set with Caco-2 cells or rat intestine. Materials and Methods: Four inhibitors of various endocytosis pathways were used [nystatin, caveolae-dependent endocytosis (CavME); dynasore, clathrin-dependent endocytosis (CME); rottlerin, macropinocytosis; and cytochalasin D, phagocytosis inhibitor], and all energy-dependent endocytosis was inhibited at temperatures under 4°C in this study. Results: Although IND-NPs showed high transintestinal penetration, no particles were detected in the basolateral side. IND-NPs penetration was strongly prevented at temperatures under 4°C. In experiments using pharmacological inhibitors, only CME inhibited penetration in the jejunum, while in the ileum, both CavME and CME significantly attenuated penetration.  Conclusion: We found a novel pathway for the transintestinal penetration of drug nanoparticles. Our hypothesis was that nanoparticles would be taken up into the intestinal epithelium by endocytosis (CME in jejunum, CavME and CME in ileum), and dissolved and diffused in the intestine. Our findings are likely to be of significant use for the development of nanomedicines.