The aim of the present study was to evaluate the cellular interaction of nanoparticles (NPs) prepared from different pegylated polymers and elucidate the effect of polymer architecture, for instance, grafted versus block copolymer on their cellular uptake. Fluorescein-labeled NPs of four different polymers, viz., poly(D,L-lactide) (PLA), poly(ethylene glycol)(1%)-graft-poly(D,L-lactide) (PEG(1%)-g-PLA), poly(ethylene glycol)(5%)-graft-poly(D,L-lactide) (PEG(5%)-g-PLA), and (poly(D,L-lactide)-block-poly(ethylene glycol)-block-poly(D,L-lactide))(n) multiblock copolymer (PLA-PEG-PLA)(n) were prepared. These NPs were characterized for their size, zeta-potential, and surface morphology. XPS studies revealed possibility of chemical interaction between PLA-COOH groups and PVA-OH groups, thus making it difficult to be washed off the NP surface completely. Grafted polymer NPs showed more surface PEG coverage than (PLA-PEG-PLA)(n) despite of their comparatively lower PEG content. The results of surface properties were translated into protein binding showing least amount of proteins bound to grafted copolymer NPs as against multiblock copolymer NPs. NPs showed no toxicity to RAW 264.7 cells. Cellular uptake of NPs was temperature and concentration-dependent as well as involved clathrin-mediated processes. Thus, this study confirms the importance of polymer architecture in determining the surface properties and hence, protein binding and cellular interactions of NPs. Also, it was shown that grafted copolymer NPs reduced macrophage uptake as compared to multiblock copolymer although mechanisms different than phagocytosis were involved.