This study aims to find the targets that may influence the production of bacitracin based on RNA sequencing in Bacillus licheniformis. Transcriptional profiling revealed that (i) the expression of the bacT gene, encoding a type II thioesterase (TEII bac ), was positively correlated with bacitracin production and (ii) the oxygen uptake exhibited significant influence on precursor synthesis. The verified experiments showed that the overexpression of TEII bac with an endogenous promoter increased the bacitracin A titer by 37.50%. Furthermore, the increase of oxygen availability through Vitreoscilla hemoglobin (VHb) expression increased the bacitracin A titer by 126.67% under oxygen-restricted conditions. From the transcriptome perspective, the results of this paper demonstrate that TEII bac and oxygen supply are crucial to bacitracin production. This study also provides insights into the construction of chassis cells for the industrial production of secondary metabolites with a preference for aerobic conditions.