Vitreoscilla hemoglobin (VHb), encoded by the Vitreoscilla hemoglobin gene (vgb), is highly effective at binding oxygen and delivering it to both prokaryotes and eukaryotes under hypoxic conditions. In this study, we introduced the vgb gene into shiitake mushrooms, and the mycelia of the transformatants grew faster. In particular, they spread into the solid substrate located in the lower part of the test tubes and bags where the oxygen was hypoxic and produced more β-glucan and plant biomass degrading enzymes compared to the original strain. The maximum growth rate of the transformants was 8.5%-15.9% higher than that of the original strain on sawdust-based cultures in plastic bags. The laccase and amylase activities were 17.7%-40.3% and 16.7%-37.9% higher than that of the original strain, respectively. In addition, the β-glucan contents of the transformant mycelia from the submerged fermentation were 12.9%-24.0% higher than that of the original strain. These results reveal that the expression of VHb in mushroom fungi promots the mycelial growth in solid-state cultures under the hypoxic condition as well as enhances β-glucan and plant biomass degrading enzymes production.