Glutathione (GSH) is the most abundant non-protein thiol in mammalian tissues, playing a critical role in antioxidant defense, detoxification of xenobiotics, and regulation of cell proliferation, apoptosis, immune function, and fibrogenesis. GSH biosynthesis occurs in the cytosol and is regulated by the availability of cysteine and the activity of glutamate cysteine ligase (GCL), which consists of GCLC (catalytic) and GCLM (modifier) subunits. GSH synthetase (GS) is the second enzyme in GSH synthesis. Key transcription factors regulating GSH synthesis include Nrf2 via the antioxidant response element (ARE), AP-1, and NFκB. Dysregulation of GSH synthesis contributes to various pathological conditions, including diabetes, liver fibrosis, alcoholic liver disease, cholestatic liver injury, endotoxemia, and drug-resistant tumors. GSH also modulates redox signaling by modifying protein cysteine residues and participates in the γ-glutamyl cycle, which allows GSH to serve as a continuous source of cysteine. GSH regulates cell growth and death by maintaining redox potential and influencing caspase activity. GSH synthesis is regulated at multiple levels, including transcriptional and post-translational regulation of GCL and GS. Factors affecting GSH synthesis include cysteine availability, transsulfuration pathway activity, and oxidative stress. GSH synthesis is dysregulated in various liver diseases, including cholestasis, endotoxemia, and fibrosis, where reduced GSH levels contribute to liver injury. Targeting GSH synthesis may offer therapeutic strategies for these conditions.Glutathione (GSH) is the most abundant non-protein thiol in mammalian tissues, playing a critical role in antioxidant defense, detoxification of xenobiotics, and regulation of cell proliferation, apoptosis, immune function, and fibrogenesis. GSH biosynthesis occurs in the cytosol and is regulated by the availability of cysteine and the activity of glutamate cysteine ligase (GCL), which consists of GCLC (catalytic) and GCLM (modifier) subunits. GSH synthetase (GS) is the second enzyme in GSH synthesis. Key transcription factors regulating GSH synthesis include Nrf2 via the antioxidant response element (ARE), AP-1, and NFκB. Dysregulation of GSH synthesis contributes to various pathological conditions, including diabetes, liver fibrosis, alcoholic liver disease, cholestatic liver injury, endotoxemia, and drug-resistant tumors. GSH also modulates redox signaling by modifying protein cysteine residues and participates in the γ-glutamyl cycle, which allows GSH to serve as a continuous source of cysteine. GSH regulates cell growth and death by maintaining redox potential and influencing caspase activity. GSH synthesis is regulated at multiple levels, including transcriptional and post-translational regulation of GCL and GS. Factors affecting GSH synthesis include cysteine availability, transsulfuration pathway activity, and oxidative stress. GSH synthesis is dysregulated in various liver diseases, including cholestasis, endotoxemia, and fibrosis, where reduced GSH levels contribute to liver injury. Targeting GSH synthesis may offer therapeutic strategies for these conditions.