Nanoparticles (NPs) have been studied for biomedical applications, ranging from prevention, diagnosis and treatment of diseases. However, the lack of the basic understanding of how NPs interact with the biological environment has severely limited their delivery efficiency to the target tissue and clinical translation. Here, we show the effective regulation of the surface properties of NPs, by controlling the surface ligand density, and their effect on serum protein adsorption, cellular uptake and cytotoxicity. The surface properties of NPs are tuned through the controlled replacement of native ligands, which favor protein adsorption, with ligands capable of increasing protein adsorption resistance. The extent and composition of the protein layer adsorbed on NPs are strongly correlated to the degree of ligands replaced on their surface and, while BSA is the most abundant protein detected, ApoE is the one whose amount is most affected by surface properties. On increasing the protein resistance, cellular uptake and cytotoxicity in mouse embryonic fibroblasts of NPs are drastically reduced, but the surface coating has no effect on the process by which NPs mainly induce cell death. Overall, this study reveals that the tuning of the surface properties of NPs allows us to regulate their biological outcomes by controlling their ability to adsorb serum proteins.