It is well known that the culture for rifamycin B production by Amycolatopsis mediterranei requires high levels of dissolved oxygen, particularly in industrial processes. In this study, we report the construction of a vector for the expression of the bacterial hemoglobin gene (vhb) from Vitreoscilla stercoraria in a rifamycin B-overproducing strain of A. mediterranei. The effect was evaluated in the presence and absence of barbital. The vhb gene was cloned under the control of the PermE promoter, the Amycolatopsis lactamdurans plasmid pULVK2 origin of replication, the kanamycin-resistant gene (Km), the erythromycin-resistant gene (ermE) for selection, and ColE1. Industrial fermentation conditions were simulated in shake-flask cultures. Under low aeration, the transformed A. mediterranei strain with the vhb gene showed a 13.9% higher production of rifamycin B in a culture with barbital compared with the parental strain, and 29.5% higher production under the same conditions without barbital.