To study charge-dependent interactions of nanoparticles (NPs) with biological media and NP uptake by cells, colloidal gold nanoparticles were modified with amphiphilic polymers to obtain NPs with identical physical properties except for the sign of the charge (negative/positive). This strategy enabled us to solely assess the influence of charge on the interactions of the NPs with proteins and cells, without interference by other effects such as different size and colloidal stability. Our study shows that the number of adsorbed human serum albumin molecules per NP was not influenced by their surface charge. Positively charged NPs were incorporated by cells to a larger extent than negatively charged ones, both in serum-free and serum-containing media. Consequently, with and without protein corona (i.e., in serum-free medium) present, NP internalization depends on the sign of charge. The uptake rate of NPs by cells was higher for positively than for negatively charged NPs. Furthermore, cytotoxicity assays revealed a higher cytotoxicity for positively charged NPs, associated with their enhanced uptake.