In this study, it was aimed to investigate characteristics and intracellular delivery of two different-sized PLGA nanoparticles in ouzo region by considering number of nanoparticles. To determine the effect of formulation parameters on average particle size, Dil labeled nanoparticles were prepared using a three-factor, two-level full factorial statistical experimental design. PLGA(230) (230.8 +/- 4.32nm) and PLGA(160) (157.9 +/- 6.16nm) nanoparticles were obtained by altering polymer amount based on experimental design results and characterized. Same number of PLGA(230) and PLGA(160) nanoparticles per cell were applied onto HEK293 cells; then, cytotoxicity, uptake kinetics and mechanism were evaluated by flow cytometry and fluorescent microscopy. Also same weight of PLGA(230) and PLGA(160) nanoparticles were applied and cellular uptake of these nanoparticles was evaluated. It was found that PLGA(230) nanoparticles had higher encapsulation efficiency and slower dye release compared to PLGA(160) nanoparticles. When they were applied at same counts per cell, PLGA(230) nanoparticles displayed faster and higher intracellular dye transfer than PLGA(160) nanoparticles. On the other hand, PLGA(160) appeared to be a more effective vehicle than PLGA(230) when applied at the same weight concentration. It was also shown that for both nanoparticles, HEK293 cells employed macropinocytic, caveolae- and clathrin-mediated endocytic pathways.