Ammonium uptake in plant roots is mediated by AMT/MEP/Rh-type ammonium transporters. Out of five AMTs being expressed in Arabidopsis roots, four AMT1-type transporters contribute to ammonium uptake, whereas no physiological function has so far been assigned to the only homolog belonging to the MEP subfamily, AMT2;1. Based on the observation that under ammonium supply, the transcript levels of AMT2;1 increased and its promoter activity shifted preferentially to the pericycle, we assessed the contribution of AMT2;1 to xylem loading. When exposed to N-labeled ammonium, amt2;1 mutant lines translocated less tracer to the shoots and contained less ammonium in the xylem sap. Moreover, in an amt1;1 amt1;2 amt1;3 amt2;1 quadruple mutant (qko), co-expression of AMT2;1 with either AMT1;2 or AMT1;3 significantly enhancedN translocation to shoots, indicating a cooperative action between AMT2;1 and AMT1 transporters. Under N deficiency, proAMT2;1-GFP lines showed enhanced promoter activity predominantly in cortical root cells, which coincided with elevated ammonium influx conferred by AMT2;1 at millimolar substrate concentrations. Our results indicate that in addition to contributing moderately to root uptake in the low-affinity range, AMT2;1 functions mainly in root-to-shoot translocation of ammonium, depending on its cell-type-specific expression in response to the plant nutritional status and to local ammonium gradients.