Several studies elucidated the transcriptional responses to iron (Fe) starvation, however, the Fe sensing and signal transduction machineries are poorly resolved thus far in plants. With this focus, by using chemical biology approach, we identified a small molecule R7, whose use inhibited the physiological and molecular responses of Fe starvation in Arabidopsis. R7 blocked the Fe starvation signaling from nitric oxide (NO) to the central transcription factor, FER-LIKE IRON-DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT). Exogenous applications of NO abduct, S-nitrosoglutathione (GSNO) were able to attenuate the R7 effect. R7 directly or indirectly decreased the endogenous GSNO levels in roots but not the NO levels. Our finding reveals that, under Fe limited situation the starvation signal from NO is passed to FIT through GSNO.