Self-assembled nanoparticles have great potential to act as vehicles for hydrophobic drug delivery. Understanding nanoparticle cellular internalization is essential for designing drugs intended for intracellular delivery. Here, the endocytosis and exocytosis of fluorescein isothiocyanate (FITC)-conjugated glycol chitosan (FGC) self-assembled nanoparticles were investigated by flow cytometry and confocal microscopy. The cellular internalization of FGC nanoparticles was initiated by nonspecific interactions between nanoparticles and cell membranes. Although adsorptive endocytosis of the nanoparticles occurred quickly, significant amounts of FGC nanoparticles were exocytosed, particularly in the early stage of endocytosis. The amount of exocytosed nanoparticles was dependent on the pre-incubation time with nanoparticles, suggesting that exocytosis is dependent on the progress of endocytosis. FGC nanoparticles internalized by adsorptive endocytosis were distributed in the cytoplasm, but not in the nucleus. In vitro cell cycle analysis demonstrated that FGC nanoparticles delivered paclitaxel into the cytoplasm and were effective in arresting cancer cell growth.