Previously, we demonstrated an important role for insulin in the protection of endothelial cells against hyperglycemic stress through maintaining cellular glutathione (GSH) redox balance. The current study focuses on the contribution of insulin to transcriptional control of endothelial cell GSH recovery during acute oxidative challenge and the influence of low glucose. The results show that insulin induced an approximate 2-fold increase in expression of gamma-glutamylcysteine ligase catalytic subunit (GCLc) mRNA and protein; interestingly, cellular GSH levels were not elevated accordingly. However, on tert-butylhydroperoxide challenge, insulin-treated cells demonstrated a robust GSH recovery that was attributed to a greater capacity for de novo synthesis via elevated GCLc levels. Notably, the effects of insulin were observed under low, but not normal, glucose conditions. Our results implicate a role for Nrf2 involvement in both constitutive and inducible endothelial GCLc expression and GSH synthesis, while PI3K/Akt/mTOR signaling appears to participate only in insulin-inducible GSH synthesis. Collectively, these results support the functional importance of insulin in Nrf2-dependent transcriptional upregulation of GCLc in GSH recovery during oxidative challenge and suggest a possible role for hypoglycemia in promoting insulin-mediated GCLc upregulation.