Mounting evidence indicate that nitric oxide (NO) acts as a signaling molecule mediating iron deficiency responses through the upregulation of the expression of iron uptake-related genes. Accordingly, NO donors such as nitrosoglutathione (GSNO) were reported to improve the fitness of plants grown under iron deficiency. Here, we showed that glutathione, a by-product of GSNO, triggered the upregulation of the expression of iron uptake- and transport-related gene and an increase of iron concentration in Arabidopsis thaliana seedlings facing iron deficiency. Furthermore, we provided evidence that under iron deficiency, NO released by GSNO did not improve the root iron concentration but impacted the content of copper. Collectively, our data highlight the complexity of interpreting data based on the use of NO donors when investigating the role of NO in iron homeostasis.