In maize, two root epidermis-expressed ammonium transporters ZmAMT1;1a and ZmAMT1;3 play major roles in high-affinity ammonium uptake.  However, the transcriptional regulation of ZmAMT1s in roots for ensuring optimal ammonium acquisition remains largely unknown.  Here, using a split root system we showed that ZmAMT1;1a and ZmAMT1;3 transcript levels were induced by localized ammonium supply to nitrogen-deficient roots.  This enhanced expression of ZmAMT1s correlated with increases in ¹⁵NH₄⁺ influx rates and tissue glutamine concentrations in roots.  When ammonium was supplied together with methionine sulfoximine, an inhibitor of glutamine synthase, ammonium-induced expression of ZmAMT1s disappeared, suggesting that glutamine rather than ammonium itself regulated ZmAMT1s expression.  When glutamine was supplied to nitrogen-deficient roots, expression levels of ZmAMT1s were enhanced, and negative feedback regulation could subsequently occur by supply of glutamine at a high level.  Thus, our results indicated an ammonium-dependent regulation of ZmAMT1s at transcript levels, and a dual role of glutamine was suggested in the regulation of ammonium uptake in maize roots.