SummaryIron is an essential micronutrient for plants and animals, and plants are a major source of iron for humans. Therefore, understanding the regulation of iron homeostasis in plants is critical. We identified a T‐DNA insertion mutant, yellow and sensitive to iron‐deficiency 1 (yid1), that was hypersensitive to iron deficiency, containing a reduced amount of iron. YID1 encodes the Arabidopsis Mediator complex subunit MED16. We demonstrated that YID1/MED16 interacted with another subunit, MED25. MED25 played an important role in regulation of iron homeostasis by interacting with EIN3 and EIL1, two transcription factors in ethylene signaling associated with regulation of iron homeostasis. We found that the transcriptome in yid1 and med25 mutants was significantly affected by iron deficiency. In particular, the transcription levels of FIT, IRT1 and FRO2 were reduced in the yid1 and med25 mutants under iron‐deficient conditions. The finding that YID1/MED16 and MED25 positively regulate iron homeostasis in Arabidopsis increases our understanding of the complex transcriptional regulation of iron homeostasis in plants.