Higher plant species differ widely in their growth responses to ammonium (NH₄⁺). However, the molecular genetic mechanisms underlying NH₄⁺ sensitivity in plants remain unknown. Here, we report that mutations in the Arabidopsis gene encoding GDP-mannose pyrophosphorylase (GMPase) essential for synthesizing GDP-mannose confer hypersensitivity to NH₄⁺. The in planta activities of WT and mutant GMPases all were inhibited by NH₄⁺, but the magnitude of the inhibition was significantly larger in the mutant. Despite the involvement of GDP-mannose in both l-ascorbic acid (AsA) and N-glycoprotein biosynthesis, defective protein glycosylation in the roots, rather than decreased AsA content, was linked to the hypersensitivity of GMPase mutants to NH₄⁺. We conclude that NH₄⁺ inhibits GMPase activity and that the level of GMPase activity regulates Arabidopsis sensitivity to NH₄⁺. Further analysis showed that defective N-glycosylation of proteins, unfolded protein response, and cell death in the roots are likely important downstream molecular events involved in the growth inhibition of Arabidopsis by NH₄⁺.