Nanoparticles are generally dispersed in electrolyte solutions for cell research. They may be aggregated and thus strongly influence the subsequent bio-effects. However, this has been often neglected in nanotoxicity research. In this paper, we selected gold nanoparticles as an example and investigated the role of dispersity in cytotoxicity. Our data indicated that the cytotoxicity of aggregated gold nanoparticles is significantly higher than well-dispersed ones. The dispersity-dependent cytotoxicity may be related to the increase of cellular endocytosis and reactive oxygen species. These results highlighted the importance of the dispersity of nanoparticles in nanotoxicity and nanobiotechnology fields.