A high concentration of ammonium (NH4+) as the sole source of nitrogen in the growth medium often is toxic to plants. The nitrate transporter NRT1.1 is involved in mediating the effects of NH4+ toxicity; however, the mechanism remains undefined. In this study, wild-type Arabidopsis (Arabidopsis thaliana Columbia-0 [Col-0]) and NRT1.1 mutants (chl1-1 and chl1-5) were grown hydroponically in NH4NO3 and (NH4)2SO4 media to assess the function of NRT1.1 in NH4+ stress responses. All the plants grew normally in medium containing mixed nitrogen sources, but Col-0 displayed more chlorosis and lower biomass and photosynthesis than the NRT1.1 mutants in (NH4)2SO4 medium. Grafting experiments between Col-0 and chl1-5 further confirmed that NH4+ toxicity is influenced by NRT1.1. In (NH4)2SO4 medium, NRT1.1 induced the expression of NH4+ transporters, increasing NH4+ uptake. Additionally, the activities of glutamine synthetase and glutamate synthetase in roots of Col-0 plants decreased and soluble sugar accumulated significantly, whereas pyruvate kinase-mediated glycolysis was not affected, all of which contributed to NH4+ accumulation. By contrast, the NRT1.1 mutants showed reduced NH4+ accumulation and enhanced NH4+ assimilation through glutamine synthetase, glutamate synthetase, and glutamate dehydrogenase. Moreover, the up-regulation of genes involved in ethylene synthesis and senescence in Col-0 plants treated with (NH4)2SO4 suggests that ethylene is involved in NH4+ toxicity responses. This study showed that NH4+ toxicity is related to a nitrate-independent signaling function of NRT1.1 in Arabidopsis, characterized by enhanced NH4+ accumulation and altered NH4+ metabolism, which stimulates ethylene synthesis, leading to plant senescence.