Abstract: In plant roots AMT-type ammonium transporters have been demonstrated to mediate high-affinity ammonium uptake across the plasma membrane. To tightly control ammonium uptake process, the regulation of AMTs at protein levels is essential. However, the underlying molecular mechanism remains unclear. Here, based on the available bioinformatics database，we predicted the putative interaction between AtAMT1;3 with AtAnkTm8 which encodes an ankyrin repeat protein. Gene expression analyses further supported this interaction because both genes were mainly expressed in the similar root tissues, and also expressed up-regulatedly under nitrogen deficiency. Two independent T-DNA insertion lines were isolated and characterized, in which the expression of AtAnkTm8 was defective. The growth of AtAnkTm8 insertion lines in the medium supplied with either ammonium as a sole nitrogen source or ammonium toxic analog methylammonium (MeA) did not differ from that of their corresponding wildtype plants. No effect on ammonium uptake capacity in AtAnkTm8 insertion lines was observed, which could be explained by the possible genetic redundancy of other AnkTm homologs. Taken together, our results describe a putative interaction between AMT and AnkTm, providing a novel regulatory mechanism on fine tuning ammonium uptake in roots.