Three hyperbranched polyglycerol nanoparticle (HPG NP) variants were synthesized and fluorescently labeled for the study of their cellular interactions. The polymeric nanoparticle that contains a hydrophobic core and a hydrophilic HPG shell, HPG-C10-HPG, is taken up faster by HT-29 cancer cells than nontransformed cells, while similar uptake rates are observed with both cell types for the nanoparticle HPG-C-10-PEG that contains a hydrophobic core and a polyethylene glycol shell. The nanoparticle HPG-104, containing neither the hydrophobic core nor the polyethylene glycol shell, is taken up faster by nontransformed cells than HT-29 cells. Importantly, cancer and normal cells can utilize different endocytic mechanisms for the internalization of these HPG NPs. Both HPG-C-10-HPG and HPG-C-10-PEG are taken up by HT-29 cells through clathrin-mediated endocytosis and macropinocytosis. Nontransformed cells, however, take up HPG-C-10-HPG and HPG-104 through macropinocytosis, while these cells utilize both clathrin-mediated endocytosis and macropinocytosis to internalize HPG-C-10-PEG.