<p>Reactive oxygen species (ROS) are endogenously generated during cellular metabolism but can also be induced by environmental stressors, such as radiation, pollutants, and inflammation. While ROS are essential for cellular function, excessive levels of ROS can inflict damage on DNA, proteins, and lipids, resulting in cellular impairment and, in severe cases, cell death. Notably, both basal ROS levels and homeostatic set point of ROS vary markedly among various subcellular compartments, with each organelle exhibiting distinct pathological consequences when its oxidative homeostasis is disrupted. Furthermore, ROS levels exhibit significant diurnal oscillations in many species, resulting in dynamic changes in cellular redox homeostasis over the 24-h cycle. Regrettably, these spatiotemporal dimensions of ROS regulation have often been overlooked in previous studies and are rarely considered in current antioxidant therapeutic strategies. This review provides a comprehensive overview of the major sites of ROS and the enzymes responsible for ROS generation and scavenging in different subcellular locations, along with their temporal variations. Additionally, the driving forces and biological functions of redox rhythms are also discussed. By integrating these insights, we aim to advance the understanding of spatiotemporal ROS regulation and provide a foundation for developing precision redox-based therapies with enhanced clinical translation.</p> Graphical Abstract <p></p>