Protein corona can alter the physiochemical properties of targeting nanoparticles (NPs), as well as their physiological responses and targeting functionality. Herein, we synthesized 20 types of NPs with diverse surface chemistry in order to study the impacts of protein corona on targeting functionality of NPs functionalized with cyclic RGD peptides and their relationships to the polyethylene glycol (PEG) length and grafting density of targeting ligands. After protein adsorption, cyclic RGD on the surface of NP was still able to bind its receptors with increased targeted cellular uptake, even at a relatively low density. However, the cellular uptake was reduced from 26 to 76% when compared with protein nonbound NPs, which was caused by the shielding effect of the outer layer adsorbed proteins. NPs functionalized with short PEG molecules and moderate cyclic RGD density performed a better targeting efficiency. Due to PEG conjugation, the protein corona was demonstrated to be beneficial for passive targeting by decreasing macrophage cellular uptake. These relationships between surface chemistry and targeting functionality will provide guidelines for the design of targeting nanoformulations in nanomedicine.