Ca2+ dynamics and oxidative signaling are fundamental mechanisms for mitochondrial bioenergetics and cell function. The MCU complex is the major pathway by which these signals are integrated in mitochondria. Whether and how these coactive elements interact with MCU have not been established. As an approach toward understanding the regulation of MCU channel by oxidative milieu, we adapted inflammatory and hypoxia models. We identified the conserved cysteine 97 (Cys-97) to be the only reactive thiol in human MCU that undergoes S-glutathionylation. Furthermore, biochemical, structural, and superresolution imaging analysis revealed that MCU oxidation promotes MCU higher order oligomer formation. Both oxidation and mutation of MCU Cys-97 exhibited persistent MCU channel activity with higher Ca2+m uptake rate, elevated mROS, and enhanced Ca2+m overload-induced cell death. In contrast, these effects were largely independent of MCU interaction with its regulators. These findings reveal a distinct functional role for Cys-97 in ROS sensing and regulation of MCU activity. Display omitted •Mitochondrial Ca2+ uniporter complex core component senses mitochondrial ROS•MCU Cys-97 undergoes S-glutathionylation and exhibits as a higher-order oligomer•Interaction between MCU and its regulatory components is unaltered by MCU oxidation•Inflammatory/hypoxia signals promote MCU oxidation and sensitize cells to death Dong et al. show that conserved MCU Cys-97 senses mitochondrial luminal ROS, and MCU oxidation promotes MCU higher-order oligomer formation and exhibits persistent activation of the MCU channel, elevated mitochondrial ROS, and enhanced Ca2+m overload-induced cell death. These findings reveal a distinct functional role for Cys-97 in mROS sensing and regulation of MCU activity.