Nanocrystals (NCs) were utilized as oral formulations in commercial products to deliver lipophilic drug, but their transport mechanisms are not fully understood. This study aimed to explore the transport mechanism of NCs using in vitro Madin-Darby canine kidney II (MDCK II) cells and in vivo larval zebrafish models. Coumarin 6 (C6) was formulated into NCs with particle size of 67.5 ± 5.2 and 190 ± 9.2 nm. In vitro studies showed that 70 nm NCs accumulated in lysosome and endoplasmic reticulum (ER) as destinations. Lipid raft pathways mediated the endocytosis, while lipid raft, ER/Golgi, and Golgi/plasma membrane pathways were involved in exocytosis and transcytosis process. However, 200 nm NCs accumulated more in a lysosome, where lipid raft pathways were also involved in the endocytosis process. In vivo studies in larval zebrafish model further confirmed that the above network plays an important role in the absorption and distribution of C6-NCs.