In order to enhance the production of validamycin A (VAL-A), a widely used agricultural antibiotic, a temperature shift strategy was developed in the fermentation of Streptomyces hygroscopicus 5008. VAL-A production and the transcriptional levels of its structural genes were enhanced in the optimal temperature shift condition. The addition of diphenyleneiodonium [DPI, reactive oxygen species (ROS) inhibitor] inhibited intracellular ROS level and VAL-A production, which indicated that ROS signal might contribute to the enhancement of VAL-A production in the temperature shift process. The transcriptional levels of stress response sigma factors SigmaB and SigmaH as well as global regulator PhoRP were enhanced, which suggested that these regulators might participate in the signal pathway. This study developed a useful strategy for VAL-A production. It will help to further understand the regulation mechanism of ROS on VAL-A synthesis. The involvement of ROS in this process will encourage researchers to develop new ROS induction strategies to enhance VAL-A production.