While ZnO particles are widely used in many fields, including personal care products, the high toxicity of ZnO nanoparticles has been reported and aroused great health concerns. In this study, the cytotoxicity of ZnO nanoparticles was evaluated and, in particular, the role of microenvironment in their toxicity was investigated. Our results show that ZnO nanoparticles are highly toxic to NIH/3T3 cells, inducing viability loss, membrane leakage and morphology changes. The microenviroment, here the CO2 atmosphere under cell culture condition, promoted the solubilization of ZnO nanoparticles. Then the released Zn from ZnO nanoparticles induces the cytotoxicity. The importance of microenvironment on the ZnO nanotoxicity is presented and the implications to future nanotoxicology studies are discussed.