Glabridin is a natural plant-derived compound that has been widely used in medicine and cosmetic applications. However, the fungicidal mechanism of glabridin against phytopathogens remains unclear. In this study, we determined the biological activity and physiological effects of glabridin against F. graminearum. Then the differentially expressed proteins of F. graminearum were screened. The EC50 values of glabridin in inhibiting the mycelial growth and conidial germination of F. graminearum were 110.70 mg/L and 40.47 mg/L respectively. Glabridin-induced cell membrane damage was indicated by morphological observations, DiBAC4(3) and PI staining, and measurements of relative conductivity, ergosterol content and respiratory rates. These assays revealed that the integrity of the membrane was destroyed, the content of ergosterol decreased, and the respiratory rate was inhibited. A proteomics analysis showed that 186 proteins were up-regulated and 195 proteins were down-regulated. Mechanically sensitive ion channel proteins related to transmembrane transport and ergosterol biosynthesis ERG4/ERG24, related to ergosterol synthesis were blocked. It is speculated that glabridin acts on ergosterol synthesis-related proteins to destroy the integrity of the cell membrane, resulting in abnormal transmembrane transport and an increased membrane potential. Finally, the morphology of mycelia was seriously deformed, growth and development were inhibited. As a result death was even induced.