Reactive oxygen species (ROS) are generated during mitochondrial oxidative metabolism and in response to xenobiotics, cytokines, and bacterial invasion. Oxidative stress, characterized by an imbalance between ROS production and cellular antioxidant defenses, can lead to macromolecular damage and various diseases such as atherosclerosis, diabetes, cancer, neurodegeneration, and aging. However, ROS also serve as critical signaling molecules in cell proliferation and survival. This review focuses on the molecular mechanisms by which ROS directly interact with critical signaling molecules to initiate signaling in a broad variety of cellular processes. The regulation of signaling pathways by ROS involves the redox regulation of cysteine residues on proteins, leading to changes in protein structure and function. Specific examples include the regulation of MAPK cascades, PI3K signaling, Nrf2-mediated redox cellular signaling, p66Shc and mitochondrial oxidative stress, and the IRE-IRP system and iron homeostasis. Additionally, ROS have been implicated in the DNA damage response through the activation of ATM and ATR kinases. Understanding the oxidative interface between ROS and signaling pathways is crucial for developing therapeutic strategies to prevent and treat ROS-mediated diseases.Reactive oxygen species (ROS) are generated during mitochondrial oxidative metabolism and in response to xenobiotics, cytokines, and bacterial invasion. Oxidative stress, characterized by an imbalance between ROS production and cellular antioxidant defenses, can lead to macromolecular damage and various diseases such as atherosclerosis, diabetes, cancer, neurodegeneration, and aging. However, ROS also serve as critical signaling molecules in cell proliferation and survival. This review focuses on the molecular mechanisms by which ROS directly interact with critical signaling molecules to initiate signaling in a broad variety of cellular processes. The regulation of signaling pathways by ROS involves the redox regulation of cysteine residues on proteins, leading to changes in protein structure and function. Specific examples include the regulation of MAPK cascades, PI3K signaling, Nrf2-mediated redox cellular signaling, p66Shc and mitochondrial oxidative stress, and the IRE-IRP system and iron homeostasis. Additionally, ROS have been implicated in the DNA damage response through the activation of ATM and ATR kinases. Understanding the oxidative interface between ROS and signaling pathways is crucial for developing therapeutic strategies to prevent and treat ROS-mediated diseases.