Reactive iron is an important pro-oxidant factor, whereas reduced glutathione (GSH) is a crucial component of a long-term adaptive system that allows cells to function during extended periods of high oxidative stress. In this work, the adaptive response of the GSH system to prolonged iron loads was characterized in human dopaminergic SH-SY5Y neuroblastoma cells. After the initial death of a substantial portion of the population, the surviving cells increased up to 5-fold their GSH content. This increase was traced to increased expression of the catalytic and the modulatory subunits of glutamate-cysteine ligase. Under conditions of high iron load, the cells maintained a low GSSG content through two mechanisms: GSSG reductase mediated recycling of GSSG to GSH, and multidrug resistant protein 1 mediated extrusion of GSSG. Increased GSH synthesis and low GSSG levels contributed to recover the cell reduction potential from -290 mV at the time of cell death to about -320 mV. These results highlight the fundamental role of GSH homeostasis in the antioxidant response to cellular iron accumulation and provide novel insights into the adaptive mechanisms of neurons subjected to increased iron loads, such as those observed in Parkinson’s disease.