NODULE INCEPTION (NIN) is a key regulator of the symbiotic nitrogen fixation pathway in legumes including Lotus japonicus. NIN-like proteins (NLPs), which are presumably present in all land plants, were recently identified as key transcription factors in nitrate signaling and responses in Arabidopsis thaliana, a non-leguminous plant. Here we show that both NIN and NLP1 of L. japonicus (LjNLP1) can bind to the nitrate-responsive cis-element (NRE) and promote transcription from an NRE-containing promoter as did the NLPs of A. thaliana (AtNLPs). However, differing from LjNLP1 and the AtNLPs that are activated by nitrate signaling through their N-terminal regions, the N-terminal region of NIN did not respond to nitrate. Thus, in the course of the evolution of NIN into a transcription factor that functions in nodulation in legumes, some mutations might arise that converted it to a nitrate-insensitive transcription factor. Because nodule formation is induced under nitrogen-deficient conditions, we speculate that the loss of the nitrate-responsiveness of NIN may be one of the evolutionary events necessary for the emergence of symbiotic nitrogen fixation in legumes.