Although ammonium (NH4+) is a key intermediate of plant nitrogen metabolism, high concentrations of NH(4)(+)in the soil provoke physiological disorders that lead to the development of stress symptoms. Ammonium nutrition was shown to induce the accumulation of glucosinolates (GSLs) in leaves of different Brassicaceae species. To further understand the link between ammonium nutrition and GSLs, we analysed the ammonium stress response of Arabidopsis mutants impaired in GSL metabolic pathway. We showed that theMYB28andMYB29double mutant (myb28myb29), which is almost deprived of aliphatic GSLs, is highly hypersensitive to ammonium nutrition. Moreover, we evidenced that the stress symptoms developed were not a consequence of the lack of aliphatic GSLs. Transcriptomic analysis highlighted the induction of an iron (Fe) deficiency response inmyb28myb29under ammonium nutrition. Consistently, ammonium-grownmyb28myb29plants showed altered Fe accumulation and homeostasis. Interestingly, we showed overall that growing Arabidopsis with increased Fe availability relieved ammonium stress symptoms and that this was associated withMYB28andMYB29expression. Taken together, our data indicated that the control of Fe homeostasis was crucial for the Arabidopsis response to ammonium nutrition and evidenced thatMYB28andMYB29play a role in this control.