NRT1.1 is a putative nitrate sensor and is involved in many nitrate-dependent responses. On the other hand, a nitrate-independent function of NRT1.1 has been implied, but the clear-cut evidence is unknown. We found that NRT1.1 mutants showed enhanced tolerance to concentrated ammonium as sole N source in Arabidopsis thaliana. This unique phenotype was not observed in mutants of NLP7, which has been suggested to play a role in the nitrate-dependent signaling pathway. Our real-time PCR analysis, and evidence from a literature survey revealed that several genes relevant to the aliphatic glucosinolate-biosynthetic pathway were regulated via a nitrate-independent signal from NRT1.1. When taken together, the present study strongly suggests the existence of a nitrate-independent function of NRT1.1.