Fast changes in environmental oxygen (O2) availability translate into shifts in mitochondrial free radical production and oxidative load that should be compensated by adequate immediate alterations in antioxidant defense mechanisms. This study explores the role hemoglobin (Hb) in oxygen-dependent modulation of reduced glutathione (GSH) levels in red blood cells. We have demonstrated that decrease in Hb oxygenation to 50% in twelve healthy humans while at high altitude, or in red blood cell suspension of heathy donors results in an increase in the intraerythrocytic GSH level that progresses with deoxygenation. Oxygen-dependent upregulation of the intracellular GSH was not associated with its de novo synthesis or release from S-glutathionylated cysteine residues of Hb molecules. Using isothermal titration calorimetry and in silico modelling, we have observed noncovalent binding of four molecules of GSH to oxy-Hb and the release of two of them from Hb upon deoxygenation and identified location of the binding sites within the Hb molecule. Oxygen-dependent binding of GSH to oxygenated Hb and its release upon deoxygenation occurs in antiphase with that of 2,3-bisphosphoglycerate. Furthermore, in the presence of GSH Hb shows increased O2 affinity. Taken together, our findings have identified an adaptive mechanism by which red blood cells may provide advanced antioxidant defense to respond to oxidative challenges immediately upon deoxygenation.