Glutathione (GSH) is the main antioxidant against cell damage. Several pathological states course with reduced nucleophilic tone and perturbation of redox homeostasis due to changes in the 2GSH/GSSG ratio. Here, we investigated the regulation of the rate-limiting GSH biosynthetic heterodimeric enzyme γ-glutamyl-cysteine ligase (GCL) by microRNAs (miRNAs). Results: "In silico" analysis of the 3′-untranslated regions (UTRs) of both catalytic (GCLc) and regulatory (GCLm) subunits of GCL enabled an identification of miR-433 as a strong candidate for the targeting of GCL. Transitory overexpression of miR-433 in human umbilical vein endothelial cells (HUVEC) showed a downregulation of both GCLc and GCLm in a nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-independent manner. Increases in pro-oxidant stimuli such as exposure to hydrogen peroxide or GSH depletion in endothelial and hepatic cells caused an expected increase in GCLc and GCLm protein expression and abrogation of miR-433 levels, thus supporting a cross-regulation of these pathways. Treatment of HUVEC with miR-433 resulted in reduced antioxidant and redox potentials, increased S-glutathionylation, and reduced endothelial nitric oxide synthase activation. In vivo models of renal and hepatic fibrosis were associated with transforming growth factor β1 (TGF-β1)-related reduction of GCLc and GCLm levels that were miR-433 dependent. Innovation and Conclusion: We describe for the first time an miRNA, miR-433, capable of directly targeting GCL and promoting functional consequences in endothelial physiology and fibrotic processes by decreasing GSH levels. Antioxid. Redox Signal. 23, 1092-1105.