In this study, we found that the zir1 mutant, defective in glutathione production, was found to be more sensitive to Fe deficiency than the wild type, and grew poorly in alkaline soil. Furthermore, the key genes involved in Fe uptake are expressed at low levels in zir1. The expression of these genes was positively correlated to the accumulation of nitric oxide (NO) under Fe deficiency. NO was proposed to be a signaling molecule involved in the up-regulation of Fe-uptake-related genes during Fe deficiency. We found that zir1 failed to accumulate NO under Fe deficiency and demonstrated that the NO mediated induction of Fe uptake genes was dependent on glutathione supply in the zir1 mutant. The results provide direct evidence that glutathione plays an essential role in Fe deficiency tolerance and NO-mediated Fe deficiency signaling.