Herein, we evaluated the cellular uptake pathways of hydrophobically modified glycol chitosan (HGC) nanoparticles as nano-sized drug carriers using cellular imaging technology. The endocytic pathway of nanocarriers for intracellular drug delivery is of great interest for the design of high efficacy delivery carriers for therapeutic agents. To evaluate the cellular uptake pathways of HGC nanoparticles, HGC was chemically labeled with near infrared (NIR) fluorescence dye, Cy5.5, to visualize the nanoparticle under confocal laser scanning microscopy. The internalization pathways of HGC nanoparticles were evaluated after treatment of specific endocytosis inhibitors. Importantly, HCG nanoparticles showed different cellular uptake efficiency and intracellular fate in cytoplasm according to the internalization pathways. Furthermore, drug distribution also evaluated according to the endocytic pathways after treatment of drug encapsulated HGC nanoparticles. As a model drug, fluorescent photosensitizer, Ce6, was encapsulated into HGC (Ce6-HGC) nanoparticles and the distribution of Ce6 in cytoplasm was evaluated using confocal laser scanning microscopy. The intracellular drug distribution showed different manner through specific endocytic pathways. The cellular imaging technology is highly useful for evaluation of endocytosis pathways and intracellular fate of drug delivery carrier which are closely related to drug distribution and therapeutic efficacy.