Hemoglobin of the Gram-negative bacterium Vitreoscilla can bind oxygen strongly and reduce the oxygen requirement of the bacterium. A recombinant plasmid pAV was constructed by inserting the hemoglobin gene (Vgb) to the downstream of the promoter of beta-lactase gene of the B. subtilis plasmid pAK4. The plasmid pAV was transferred into strain DB104, G331 (a subtilisin-producing B. subtilis) and B53 (a xylanase-producing B. subtilis). The subclones and transfer of the Vgb gene into pAK4 were detected and confirmed by Dot blotting and electrophoresis analysis. The expression of homoglobin was measured by recording CO-difference spectra after bubbling CO into the sample cuvette. The production of subtilisin and xylanase was increased in the fermentation of the Vgb-expressing strains. These results provided a new way to reduce the oxygen requirement and to increase the biomass production in the fermentation industry.