Primers for PCR cloning were designed according to periplasmic hemin- binding protein(HutB)gene sequence of Vibrio alginolyticus published in GenBank. The hutB gene of V.alginolyticus strain HY9901 was amplified by PCR and cloned into pMD18-T vector. Sequence analysis revealed that hutB gene is 870 bp and encodes a putative protein of 289 amino acids. The predicted molecular weight (MW) of HutB was 30.59 kD with an estimated pI of 6.45. Using SignalP 4.0 and TMHMM Server 2.0 software, it was predicted that the HutB protein was located in periplasmic. It contained a signal peptide cutting site, but did not have transmembranous region. This protein had one cAMP- and cGMP- dependent protein kinase phosphorylation site, four protein kinase C phosphorylation sites, and so on. To further analyze the evolutionary relationship among HutB, a molecular phylogenetic tree was constructed using Mega 5.0 software. In this tree, the HutB protein showed high genetic relationship with Vibrio campbellii and Vibrio harveyi. Using qRT-PCR, the expression changes of hutB under various iron sources were examed. Cultured in iron-rich medium containing FeCl₃, the expression levels of hutB changed a little compared with the control group. However, in the medium with hemin, the expression level of hutB increased, specifically in the iron-restricted environment exists with hemin, the expression of hutB increased significantly(P<0.01). At the same time, the expression of hutB under iron limiting environment with: 2,2'-Dipyridyl was lower than control group(P<0.01).