Abstract: Ferroptosis is a regulated cell death modality driven by iron-dependent lipid peroxide accumulation, and is involved in ischemia-reperfusion injury, neurodegenerative diseases, and cancer therapy. The cysteine/glutathione/glutathione peroxidase 4 (Cys/GSH/GPX4) axis is central to defense against ferroptosis. This review systematically summarizes the structural basis of GPX4, the localization and functions of its three isoforms, and the multilevel regulatory network comprising transcriptional/epigenetic regulation, translation, post-translational modifications, and autophagy/proteasome-mediated degradation. Key findings indicate that GPX4 exhibits cell type and microenvironment-dependent bidirectional roles in cancer, cardiovascular diseases, neurodegenerative diseases, sepsis, inflammatory bowel disease, rheumatoid arthritis, and systemic lupus erythematosus, protecting normal cells while contributing to tumor drug resistance and immune evasion. Therapeutically, covalent/allosteric inhibitors, proteolysis-targeting chimera (PROTAC)/autophagy-targeting chimera (AUTAC) degraders, nanodelivery, and combinations with radiotherapy/chemotherapy/immunotherapy show potential. This review proposes that GPX4 is a key hub linking redox homeostasis and therapeutic resistance. Future efforts should focus on constructing a complete regulatory atlas and developing isoform-specific probes, degraders, and smart delivery systems for precise targeting.