Reactive oxygen species (ROS) are generated during mitochondrial oxidative metabolism and in response to xenobiotics, cytokines, and bacterial invasion. Oxidative stress occurs when ROS levels exceed the cell's antioxidant capacity, leading to macromolecular damage and contributing to diseases like atherosclerosis, diabetes, cancer, neurodegeneration, and aging. However, ROS also serve as critical signaling molecules in cell proliferation and survival. This review explores how ROS directly regulate signaling molecules, termed the "oxidative interface," in various cellular processes, including proliferation, survival, ROS homeostasis, antioxidant gene regulation, mitochondrial oxidative stress, apoptosis, aging, iron homeostasis, and DNA damage response. ROS regulate signaling pathways through redox modifications of cysteine residues on proteins, altering their structure and function. For example, ROS activate the MAPK pathways by oxidizing and activating ASK1, which in turn phosphorylates and activates JNK and p38 MAPKs. ROS also regulate PI3K signaling by modulating PTEN activity, which is crucial for maintaining cellular homeostasis. The Nrf2 pathway is central to antioxidant gene regulation, with ROS-induced redox changes in Nrf2 leading to its nuclear translocation and activation of antioxidant genes. ROS also influence iron homeostasis through the IRE-IRP system, where redox modifications of IRP1 and IRP2 regulate iron transport and storage. ROS play a key role in DNA damage response by activating ATM, which is crucial for cellular defense against oxidative stress. ROS can directly activate ATM through disulfide bond formation, leading to downstream signaling events that promote DNA repair and cell survival. Overall, ROS are essential for both harmful and beneficial cellular processes, highlighting the importance of understanding their redox regulation in disease prevention and therapy.Reactive oxygen species (ROS) are generated during mitochondrial oxidative metabolism and in response to xenobiotics, cytokines, and bacterial invasion. Oxidative stress occurs when ROS levels exceed the cell's antioxidant capacity, leading to macromolecular damage and contributing to diseases like atherosclerosis, diabetes, cancer, neurodegeneration, and aging. However, ROS also serve as critical signaling molecules in cell proliferation and survival. This review explores how ROS directly regulate signaling molecules, termed the "oxidative interface," in various cellular processes, including proliferation, survival, ROS homeostasis, antioxidant gene regulation, mitochondrial oxidative stress, apoptosis, aging, iron homeostasis, and DNA damage response. ROS regulate signaling pathways through redox modifications of cysteine residues on proteins, altering their structure and function. For example, ROS activate the MAPK pathways by oxidizing and activating ASK1, which in turn phosphorylates and activates JNK and p38 MAPKs. ROS also regulate PI3K signaling by modulating PTEN activity, which is crucial for maintaining cellular homeostasis. The Nrf2 pathway is central to antioxidant gene regulation, with ROS-induced redox changes in Nrf2 leading to its nuclear translocation and activation of antioxidant genes. ROS also influence iron homeostasis through the IRE-IRP system, where redox modifications of IRP1 and IRP2 regulate iron transport and storage. ROS play a key role in DNA damage response by activating ATM, which is crucial for cellular defense against oxidative stress. ROS can directly activate ATM through disulfide bond formation, leading to downstream signaling events that promote DNA repair and cell survival. Overall, ROS are essential for both harmful and beneficial cellular processes, highlighting the importance of understanding their redox regulation in disease prevention and therapy.