The effects of reactive oxygen species (ROS) on cell growth and poly (γ-glutamic acid) (γ-PGA) synthesis were studied by adding hydrogen peroxide (H2O2) to the medium of Bacillus subtilis NX-2. After optimizing the addition concentration and time of H2O2, a maximum concentration of 33.9 g/L γ-PGA was acquired by adding 1% (v/v) H2O2 to the medium at 24th hour, which was 20.6 % higher than that of the control. The addition of diphenyleneiodonium chloride (ROS inhibitor) could interdict the effect of H2O2-induced ROS. Furthermore, changes in the intracellular nucleotide levels and transcriptional levels were investigated in the batch fermentation under ROS stress. A lower NADH/NAD+ ratio and higher ATP levels were achieved, which could provide more reducing power and energy for γ-PGA biosynthesis. The transcriptional levels of the genes belonging to the tricarboxylic acid (TCA) cycle, electron transfer chain (ETC) system and γ-PGA synthesis pathways noticeably increased, which revealed that ROS regulated the γ-PGA synthesis process as a signal molecule on the transcriptional level. These results indicated that the γ-PGA synthesis pathway was enhanced by ROS from the perspective of energy metabolism and genetic transcription.