[Background] Maize sheath blight has grew into the main disease that restricted the increase in maize production in China. Validamycin A and biocontrol microbe Trichoderma sp. both have resistance to sheath blight but each of which provides benefits and drawbacks. [Objective] To study the synergy of Validamycin A and Trichoderma for the control of sheath blight of maize. [Methods] We determined the effect of Validamycin A on cell wall degradation enzymes and related enzyme in defense reaction of Trichoderma asperellum GDSF1009. We used tobacco leaves to validate reactive oxygen and its allergic reactions. We also used the transcriptome and gas chromatography-time of flight-mass spectrometry to analyze Validamycin A’s effect on the expression difference of genes, the growth and the metabolism of GDSF1009 and carried out the experiment on the synergistic inhibition of leaf and plate. [Results] Validamycin A had no effect on the activities of chitinase, cellulase and xylanase of GDSF1009 and did not get into the cell. At the level of transcriptome, compared with the controls, the proportions of different genes in Trichoderma asperellum were 8.932% and 6.779% respectively after 24 h and 48 h treated by Validamycin A. Validamycin A had no significant influence on carbohydrate and lipid and only had some influences on genes related to amino acid metabolism. These genes would not change amino acid metabolism much. Furthermore, the combination of the Validamycin A and Trichoderma asperellum can significantly improve the effect of prevention of maize sheath blight. [Conclusion] Validamycin A is safe for the primary metabolic system of Trichoderma asperellum. The resistance of the synergy is significantly higher than that of the single factor.