Tuning the bandgap and cytotoxicity of ZnO nanoparticles (NPs) is very important, not only for customizing their optoelectronic and biomedical applications, but also for their cytotoxicity assay and safe usage. A unique soft-template of polyvinylpyrrolidone has been developed here to realize a rapid room-temperature neutral synthesis of ZnO with controlled nanostructures for tuning the bandgap and cytotoxicity of ZnO. By simply changing the reagent stoichiometry and the soft-template shape, high-purity ZnO rods, tripods, tubes, and unique T-like tubes with tunable size, surface composition/charge, bandgap, and cytotoxicity are obtained. It has been revealed that the ZnO bandgap can be remarkably reduced by introducing the surface nonstoichiometry; and the ZnO-induced cytotoxicity can be tuned by the size, shape, surface charge/composition, and bandgap of ZnO NPs at different degrees. Significantly, both the photochemistry reaction and the reactive oxygen species induced by ZnO NPs are not necessary for the ZnO-induced cytotoxicity.