Poly gamma glutamic acid (-PGA) is an anionic polyamide with numerous applications. Previous studies revealed that L-proline metabolism is implicated in a wide range of cellular processes by increasing intercellular reactive oxygen species (ROS) generation. However, the relationship between L-proline metabolism and -PGA synthesis has not yet been analyzed. In this study, our results confirmed that deletion of 1-pyrroline-5-carboxylate dehydrogenase gene ycgN in Bacillus licheniformis WX-02 increased -PGA yield to 13.91gL(-1), 85.22% higher than that of the wild type (7.51gL(-1)). However, deletion of proline dehydrogenase gene ycgM had no effect on -PGA synthesis. Furthermore, a 2.92-fold higher P5C content (19.24molgDCW(-1)) was detected in the ycgN deficient strain WXycgN, while the P5C levels of WXycgM and the double mutant strain WXycgMN showed no difference, compared to WX-02. Moreover, the ROS level of WXycgN was increased by 1.18-fold, and addition of n-acetylcysteine (antioxidant) decreased its ROS level, which further reduced -PGA synthesis capability of WXycgN. Collectively, our results demonstrated that proline catabolism played an important role in maintaining ROS homeostasis, and deletion of ycgN-enhanced P5C accumulation, which induced a transient ROS signal to promote -PGA synthesis in B. licheniformis.