Glutamate waste liquid is the waste produced in the production process of glutamic acid, with low pH, high ammonium, and high sulfate. The waste liquid contains glutamic acid and can be used as a raw material to produce poly-γ-glutamic acid (γ-PGA), achieving the recycling of waste liquid. [Objective] To investigate the inhibitory effect of glutamate waste liquid on γ-PGA synthesis, we used Bacillus subtilis KH2 to synthesize γ-PGA and evaluated the inhibitory effect of glutamate waste liquid on the synthesis of γ-PGA. [Methods] Comparative transcriptomics was employed to excavate the key genes and inhibitory factors involved in γ-PGA synthesis, and key gene overexpression and knockout were conducted to identify the inhibitory factors. Fermentation experiments were then performed for verification. [Results] The glutamate waste liquid as the substrate for production of γ-PGA by fermentation showed significant inhibitory effects. A total of 1 819 significantly differentially expressed genes were identified, including 952 genes with significantly up-regulated expression and 867 genes with significantly down-regulated expression. The transcript levels of 10 genes (alsS, pgsA, gltT, budA, fumC, ptsG, racE, opuAB, acoC, and rocG) involved in γ-PGA synthesis of B. subtilis KH2 changed significantly during primary fermentation and glutamate waste liquid fermentation. Eight down-regulated genes (alsS, pgsA, gltT, budA, fumC, ptsG, racE, and opuAB) were overexpressed, which increased the production of γ-PGA by 91.20%, 120.77%, 137.50%, 36.44%, 40.85%, 104.58%, 65.67%, and 69.72%, respectively. The overexpression of pgsA, gltT, ptsG, racE, and opuAB increased glutamic acid utilization by 11.57%, 35.53%, 12.83%, 21.43%, and 14.80%, respectively. The overexpression of alsS, budA, and fumC had no obvious improving effect on the utilization of glutamic acid. The knockout of two up-regulated genes (acoC and rocG) had little effect on γ-PGA production and glutamic acid utilization. [Conclusion] The downregulation of ptsG, gltT, racE, pgsA, and fumC in waste liquid fermentation has significant effects on substrate utilization, glutamic acid configuration conversion and polymerization, and TCA cycle, which reduces the synthesis efficiency of γ-PGA. This study reveals the inhibitory mechanism of glutamate waste liquid in γ-PGA synthesis and provides a sustainable biotechnology for the production of value-added biopolymers from industrial waste liquid.