Ammonium (NH4+) is a key inorganic nitrogen source in cellular amino acid biosynthesis. The coupling of transcriptional and posttranslational regulation of AMMONIUM TRANSPORTER (AMT) ensures that NH4+ acquisition by plant roots is properly balanced, which allows for rapid adaptation to a variety of nitrogen conditions. Here, we report that phospholipase D (PLD)-derived phosphatidic acid (PA) interacts with AMT1; 1 to mediate NH4+ uptake in Arabidopsis (Arabidopsis thaliana). We examined pldα1 pldδ-knockout mutants and found that a reduced PA level increased seedling growth under nitrogen deficiency and inhibited root growth upon NH4+ stress, which was consistent with the enhanced accumulation of cellular NH4+. PA directly bound to AMT1; 1 and inhibited its transport activity. Mutation of AMT1; 1 R487 to Gly (R487G) resulted in abolition of PA suppression and, subsequently, enhancement of ammonium transport activity in vitro and in vivo. Observations of AMT1; 1-GFP showed suppressed endocytosis under PLD deficiency or by mutation of the PA-binding site in AMT1; 1. Endocytosis was rescued by PA in the pldα1 pldδ mutant but not in the mutant AMT1; 1R487G-GFP line. Together, these findings demonstrated PA-based shutoff control of plant NH4+ transport and point to a broader paradigm of lipid-transporter function.