Herein, we demonstrate that the surface charge of gold nanoparticles (AuNPs) plays a critical role in modulating membrane potential of different malignant and nonmalignant cell types and subsequent downstream intracellular events. The findings presented here describe a novel mechanism for cell-nanoparticle interactions and AuNP uptake: modulation of membrane potential and its effect on intracellular events. These studies will help understand the biology of cell-nanoparticle interactions and facilitate the engineering of nanoparticles for specific intracellular targets.