Glutathione biosynthesis is a key component in the network of plant stress responses that counteract oxidative damage and maintain intracellular redox environment. Using a combination of mass spectrometry and site-directed mutagenesis, we examined the response of Arabidopsis thaliana glutamate-cysteine ligase (GCL) to changes in redox environment. Mass spectrometry identified two disulfide bonds (Cys186-Cys406 and Cys349-Cys364) in GCL. Mutation of either Cys-349 or Cys-364 to a Ser reduced reaction rate by twofold, but substitution of a Ser for either Cys-186 or Cys-406 decreased activity by 20-fold and abrogated the response to changes in redox environment. Redox titrations show that the regulatory disulfide bond has a midpoint potential comparable with other known redox-responsive plant proteins. Mutation of Cys-102, Cys-251, Cys-349, or Cys-364 did not alter the response to redox environment, indicating that modulation of activity depends on the Cys186-Cys406 disulfide bond. In vivo analysis of GCL in Arabidopsis root extracts revealed that multiple oxidative stresses altered the distribution of oxidized (active) and reduced (inactive) enzyme and that this change correlated with increased GCL activity. The thiol-based regulation of GCL provides a posttranslational mechanism for modulating enzyme activity in response to in vivo redox environment and suggests a role for oxidative signaling in the maintenance of glutathione homeostasis in plants.