The effect of ethanol on the production of the important agro-antibiotic validamycin A (Val-A) in medium containing agricultural by-products was investigated. Under the optimal condition of ethanol addition, the maximal Val-A production titer reached 18 g/L, which increased by 60% compared to the control. To provide an insight into cell response to ethanol, the intracellular reactive oxygen species (ROS), gene transcription and enzyme activity were determined. Intracellular ROS as the molecular signal was increased in the ethanol condition. Global regulators afsR and glnR were involved in regulation of Val-A biosynthesis, and the transcription of eight Val-A structural genes was enhanced. The activity of glucose-6-phosphate dehydrogenase (G6PD) was enhanced while glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was inhibited. A signal transduction cascade from cell signal response to activated transcription of Val-A biosynthetic genes and enhanced antibiotic production is proposed. The information can be helpful for the improvement of large-scale fermentation. (C) 2012 Elsevier Ltd. All rights reserved.