Self-microemulsifying drug delivery systems (SMEDDSs) have recently returned to the limelight of academia and industry due to their enormous potential in oral delivery of biomacromolecules. However, information on gastrointestinal lipolysis and trans-epithelial transport of SMEDDS is rare. Aggregation-caused quenching (ACQ) fluorescent probes are utilized to visualize the behaviors of SMEDDSs, because the released probes during lipolysis are quenched upon contacting water. Two SMEDDSs composed of medium chain triglyceride and different ratios of Tween-80 and PEG-400 are set as models, meanwhile Neoral® was used as a control. The SMEDDS droplets reside in the digestive tract for as long as 24 h and obey first order kinetic law of lipolysis. The increased chain length of the triglyceride decreases the lipolysis of the SMEDDSs. imaging of main tissues and histological examination confirm the trans-epithelial transportation of the SMEDDS droplets. Approximately 2%-4% of the given SMEDDSs are transported the lymph route following epithelial uptake, while liver is the main termination. Caco-2 cell lines confirm the cellular uptake and trans-epithelial transport. In conclusion, a fraction of SMEDDSs can survive the lipolysis in the gastrointestinal tract, permeate across the epithelia, translocate the lymph, and accumulate mainly in the liver.