In plant, iron uptake and homeostasis are tightly regulated to ensure its absorption from soil and to avoid excess iron in the cell. Many genes involved in this process have been identified during past several years, but there are many problems remain unsolved in the genetic regulation of whole plant iron trafficking and allocation. MYB transcription factors contain tandem repeats of a approximately 50 amino acid DNA-binding motif (R) and are involved in the regulation of many aspects of plant development, hormone signaling and metabolism. Here, we report that the ectopic expression of orchid R2R3-MYB gene DwMYB2 in Arabidopsis thaliana confers the transgenic plants hypersensitivity to iron deficiency. In DwMYB2 transgenic plants, the iron content in root is two-fold higher compared to that in wild-type root, while the reverse is true in shoot. This imbalance of iron content in root and shoot suggested that the translocation of iron from root to shoot was affected by the expression of DwMYB2 in the transgenic plants. Consistently, gene chip and reverse transcription-polymerase chain reaction analysis revealed that the ferric-chelate reductase gene, AtFRO2, and the iron transporter gene, AtIRT1 and AtIRT2, are up-regulated by DwMYB2 expression, while other potential iron transporters such as AtIREG1, AtFRD3 and NRAMP1 are down-regulated. In addition, the expression of several putative peptide transporters and transcription factors are also altered in the 35S::DwMYB2 transgenic lines. These data provide us insight into the whole plant translocation of iron and identify candidate genes for iron homeostasis in plants despite the fact that a heterologous gene was expressed.