Abstract Background Ammonium (NH4 +), a key nitrogen form, becomes toxic when it accumulates to high levels. Ammonium transporters (AMTs) are the key transporters responsible for NH4 + uptake. AMT activity is under allosteric feedback control, mediated by phosphorylation of a threonine in the cytosolic C-terminus (CCT). However, the kinases responsible for the NH4 +-triggered phosphorylation remain unknown. Results In this study, a functional screen identified protein kinase CBL-Interacting Protein Kinase15 (CIPK15) as a negative regulator of AMT1;1 activity. CIPK15 was able to interact with several AMT1 paralogs at the plasma membrane. Analysis of AmTryoshka, an NH4 + transporter activity sensor for AMT1;3 in yeast, and a two-electrode-voltage-clamp (TEVC) of AMT1;1 in Xenopus oocytes showed that CIPK15 inhibits AMT activity. CIPK15 transcript levels increased when seedlings were exposed to elevated NH4 + levels. Notably, cipk15 knockout mutants showed higher 15NH4 + uptake and accumulated higher amounts of NH4 + compared to the wild-type. Consistently, cipk15 was hypersensitive to both NH4 + and methylammonium but not nitrate (NO3 −). Conclusion Taken together, our data indicate that feedback inhibition of AMT1 activity is mediated by the protein kinase CIPK15 via phosphorylation of residues in the CCT to reduce NH4 +-accumulation.