Although some formulations based on nanotechnology are already available, the transport of nanoscale particles including polymeric micelles (PMs) across epithelial cell monolayer was barely studied. To prove the transport of PMs across the epithelial barrier and explain the mechanisms related, a typical PM system containing a fluorescent probe Coumarin 6 (C6) was prepared and the Madin–Darby canine kidney (MDCK) cell line was used as an epithelial cell model. Four different approaches were applied to monitor the transport of PMs prepared, including the real-time and in situ imaging by a novel approach developed by our group. And the solid evidence of PMs' transport was obtained. The mechanism related was explored by different techniques. With the absence of caveolae mediated endocytosis and macropinocytosis, the clathrin mediated pathway might play a great role here, and a fraction of PMs bypassed the degradative lysosome pathway, probably due to the clathrin and caveolae independent mechanisms. Interestingly, some inhibitor was found to inhibit transcytosis of PMs significantly but not their endocytosis, suggesting different mechanisms involved in endocytosis and exocytosis. The Forster resonance energy transfer (FRET) phenomenon still existed after FRET PMs were internalized by cells. Anyhow, a multiple process with active transcellular pathway was indicated.