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Potential biomedicinal applications of graphene oxide (GO), for example, as a carrier of biomolecules or a reagent for photothermal therapy and biosensing, are limited by its cytotoxicity and mutagenicity. It is believed that these properties are at least partially caused by GO‐induced oxidative stress in cells. However, it is not known which chemical fragments of GO are responsible for this unfavorable effect. We generated four GOs containing variable redox‐active groups on the surface, including Mn²⁺, C‐centered radicals, and endoperoxides (EPs). A comparison of the abilities of these materials to generate reactive oxygen species in human cervical cancer cells revealed that EPs play a crucial role in GO‐induced oxidative stress. These data could be applied to the rational design of biocompatible nontoxic GOs for biomedical applications.