Regulation of iron homeostasis in maize remains unclear, despite the known roles of FER-Like Fe deficiency-induced transcription factor (FIT) in Arabidopsis and rice. ZmFIT, like AtFIT and OsFIT, interacts with iron-related transcription factors 2 (ZmIRO2). Here, we investigate the involvement of ZmFIT in iron homeostasis. Mutant ZmFIT lines exhibiting symptoms of Fe deficiency had reduced shoot iron content. Transcriptome analysis revealed downregulation of Fe deficiency-responsive genes in the roots of a Zmfit mutant. ZmFIT facilitates the nuclear translocation of ZmIRO2 to activate transcription of downstream genes under Fe-deficient conditions. Our findings suggest that ZmFIT, by interaction with ZmIRO2, mediates iron homeostasis in maize. Notably, the binding and activation mechanisms of ZmFIT resemble those in Arabidopsis but differ from those in rice, whereas downstream genes regulated by ZmFIT show similarities to rice but differences from Arabidopsis. In brief, ZmFIT, orthgologs of OsFIT and AtFIT in rice and maize, respectively, regulates iron uptake and homeostasis in maize, but with variations.