When iron is scarce, Schizosaccharomyces pombe cells repress transcription of several genes that encode iron-using proteins. Php4 mediates this transcriptional control by specifically interacting with the CCAAT-binding core complex that is composed of Php2, Php3, and Php5. In contrast, when there is sufficient iron, Php4 is inactivated, thus allowing the transcription of many genes that encode iron-requiring proteins. Analysis by bimolecular fluorescence complementation and two-hybrid assays showed that Php4 and the monothiol glutaredoxin Grx4 physically interact with each other. Deletion mapping analysis revealed that the glutaredoxin (GRX) domain of Grx4 associates with Php4 in an iron-dependent manner. Site-directed mutagenesis identified the Cys172 of Grx4 as being required for this iron-dependent association. Subsequent analysis showed that, although the thioredoxin (TRX) domain of Grx4 interacts strongly with Php4, this interaction is insensitive to iron. Fine mapping analysis revealed that the Cys35 of Grx4 is necessary for the association between the TRX domain and Php4. Taken together, the results revealed that whereas the TRX domain interacts constitutively with Php4, the GRX domain-Php4 association is both modulated by iron and required for the inhibition of Php4 activity in response to iron repletion.