Vibrio tubiashii expresses virulence factors, such as a vulnificolysin-like hemolysin or cytolysin and a zinc metalloprotease, similar to those of other pathogenic vibrios. In this study, we report the cloning of a novel hemolysin gene of V. tubiashii in Escherichia coli . A V. tubiashii gene library was screened for hemolytic activity on sheep blood agar. Three hemolytic clones pGem:hly1, pGem:hly2, and pGem:hly3 were sequenced, and the sequences showed a strong homology to the ribA gene coding for guanosine triphosphate cyclohydrolase II (GCH II), required for riboflavin biosynthesis and reported to be responsible for hemolytic activity in Helicobacter pylori . The plasmids pGem:hly1 and pGem:hly3 when introduced into E. coli BSV18 (ribA18::Tn5) were able to restore growth of strain BSV18 in a medium without riboflavin and also produced hemolytic activity on blood agar. PCR primers based on the cloned hly-ribA sequence were tested using 23 different Vibrio strains representing 10 different species. Amplification of ribA gene locus only occurred with V. tubiashii strains. In summary, our results indicate that we have cloned a ribA homolog of V. tubiashii that imparts hemolytic activity to E. coli clones, and primers based on this gene locus might be useful as a species-specific identification tool for V. tubiashii.