BACKGROUND: Iron (Fe) is the most limiting micronutrient element for crop production in alkaline soils. A number of transcription factors involved in regulating Fe uptake from soil and transport in plants have been identified. Analysis of transcriptome data from Oryza sativa grown under limiting Fe conditions reveals that transcript abundances of several genes encoding transcription factors are altered by Fe availability. These transcription factors are putative regulators of Fe deficiency responses. RESULTS: Transcript abundance of one nuclear located basic helix-loop-helix family transcription factor, OsIRO3, is up-regulated from 25- to 90-fold under Fe deficiency in both root and shoot respectively. The expression of OsIRO3 is specifically induced by Fe deficiency, and not by other micronutrient deficiencies. Transgenic rice plants over-expressing OsIRO3 were hypersensitive to Fe deficiency, indicating that the Fe deficiency response was compromised. Furthermore, the Fe concentration in shoots of transgenic rice plants over-expressing OsIRO3 was less than that in wild-type plants. Analysis of the transcript abundances of genes normally induced by Fe deficiency in OsIRO3 over-expressing plants indicated their induction was markedly suppressed. CONCLUSION: A novel Fe regulated bHLH transcription factor (OsIRO3) that plays an important role for Fe homeostasis in rice was identified. The inhibitory effect of OsIRO3 over-expression on Fe deficiency response gene expression combined with hypersensitivity of OsIRO3 over-expression lines to low Fe suggest that OsIRO3 is a negative regulator of the Fe deficiency response in rice.