Under physiological conditions, cells are in a stable state known as redox homeostasis, which is maintained by the balance between continuous ROS/RNS generation and several mechanisms involved in antioxidant activity. ROS overproduction results in alterations in the redox homeostasis that promote oxidative damage to major components of the cell, including the biomembrane phospholipids. Lipid peroxidation subsequently generates a diverse set of products, including α,β-unsaturated aldehydes. Of these products, 4-hydroxy-2-nonenal (HNE) is the most studied aldehyde on the basis of its involvement in cellular physiology and pathology. This review summarizes the current knowledge in the field of HNE generation, metabolism, and detoxification, as well as its interactions with various cellular macromolecules (protein, phospholipid, and nucleic acid). The formation of HNE-protein adducts enables HNE to participate in multi-step regulation of cellular metabolic pathways that include signaling and transcription of antioxidant enzymes, pro-inflammatory factors, and anti-apoptotic proteins. The most widely described roles for HNE in the signaling pathways are associated with its activation of kinases, as well as transcription factors that are responsible for redox homeostasis (Ref-1, Nrf2, p53, NFκB, and Hsf1). Depending on its level, HNE exerts harmful or protective effects associated with the induction of antioxidant defense mechanisms. These effects make HNE a key player in maintaining redox homeostasis, as well as producing imbalances in this system that participate in aging and the development of pathological conditions.