The effect of the coating polymer poly(N-vinyl-pyrrolidone) (PVP) on the protein adsorption, phagocytosis, and pharmacokinetics of poly(D,L-lactide)-based nanoparticles was evaluated in vitro and in vivo. Control poly(ethylene glycol) (PEG)-coated nanoparticles were included for comparison. While no difference between PEG- and PVP-decorated nanoparticles in terms of amount of adsorbed protein was evident upon incubation in single protein solutions (BSA, IgG), incubation in serum revealed a protein adsorption pattern both quantitatively and qualitatively distinct. Larger amounts of complement components and immunoglobulins were found to adhere to PVP-coated particles, whereas PEG particles showed preferential adsorption of apolipoproteins. Furthermore, preopsonization in fresh rather than heat-inactivated serum enhanced uptake of both types of particles by murine RAW 264.7 macrophages. However, when isolated rat Kupffer cells were employed, activation of the complement system significantly enhanced the uptake of PVP-coated nanoparticles compared to PEG particles. Ultimately, PVP-coated nanoparticles exhibited considerably shorter circulation times compared to their PEG counterparts when administered intravenously to rats.