Hemoglobins (Hbs) are heme proteins found in all five kingdoms of living organisms. In plants, three different classes of Hbs have been identified-nonsymbiotic Hbs from diverse species, symbiotic Hbs from nitrogen-fixing plants, and so-called 2-on-2 Hbs. Here, we report the cloning and expression analysis of the 2-on-2 Hb gene, GmGLB3, from soybean. The GmGLB3 cDNA clone encodes a protein for 172 amino acid residues. Its deduced amino acid sequence shows the highest identity (74%) with 2-on-2 Hb from Medicago truncatula. Multiple sequence alignment confirms the conserved and signature amino acid residues previously reported with plant 2-on-2 Hbs. Genomic Southern hybridization demonstrates that GmGLB3 has two copies in the soybean genome. Based on our northern hybridization, the GmGLB3 gene is specifically expressed in root nodules, with levels increasing in the late stage during nodule development. Its transcript level is also increased under flooding and kinetin treatments in the roots, or under flooding and 2-iP treatments in the stems. However, no transcript is detected in the leaves regardless of treatment. Therefore, we propose that the GmGLB3 gene is specifically expressed in root nodules and that its expression in other plant organs is regulated by cytokinin and/or flooding.