Pneumocandin B-0, the precursor of the antifungal drug caspofungin, is a lipohexapeptide produced by the fungus Glarea lozoyensis. Oxidative stress and the resulting production of reactive oxygen species (ROS) are known to be involved in the regulation of pneumocandin B-0 biosynthesis. In this study, the Glyap1 gene of Glarea lozoyensis, a homologue of the yeast redox regulator YAP1, was knocked out. The intracellular ROS levels of the resulting Delta Glyap1 strain were higher than in the wild-type strain, which was caused by the downregulated expression of superoxide dismutase (SOD) and catalase (CAT). Compared with the wild-type strain, Delta Glyap1 exhibited an oxidative phenotype throughout its life cycle, which resulted in significantly higher pneumocandin B-0 production per unit biomass. In addition, Delta Glyap1 showed growth inhibition and decreased pneumocandin B-0 production in the presence of CCl4, which leads to strong oxidative stress. To overcome the strain's sensitivity, a three-stage antioxidant addition strategy was developed. This approach significantly improved the growth of Delta Glyap1 while maintaining a high pneumocandin B-0 production per unit biomass, which reached 38.78 mg/g DCW. Notably, this result represents a 50% increase over the wild-type strain. These findings provide new insights into the regulatory mechanisms that control pneumocandin B-0 production under oxidative stress, which may be applied to improve the production of other secondary metabolites.