Excessive amounts of nickel (Ni) can be toxic for plants. Recently, we reported that IRT1, the primary iron (Fe) uptake transporter in roots, meditates excess Ni accumulation in Arabidopsis thaliana. We also found that Ni exposure increases IRT1 expression in roots, suggesting that Ni uptake is further induced by Ni stress. Here, we show that Ni exposure induces expression of not only IRT1, but also FRO2, a ferric reductase in the root epidermis, and FIT, a transcription factor regulating the expression of genes involved in Fe homeostasis including IRT1 and FRO2. This result suggests that Ni accumulation induces an Fe-deficient response and leads to the induction of IRT1. Our findings suggest that excess Ni causes Fe deficiency at the molecular level and induces Fe deficiency signaling in plant cells.