Abstract:[Objective] Endophytic fungi of medicine plants are the new microbial resource of antibiotic medicines. These antibacterial mechanisms of endophytic fungi named JY25 from Gynostemma pentaphyllum have important significance for the research and development of endophytic fungi. [Methods] The method of double dilution has adopted to measure the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of fermentation broth to Escherichia coli. Then, the JY25 fermentation broth with MIC concentrations was used to inhibit the growth of E. coli, and the morphology changes of E. coli were observed by scanning electron microscope. Meanwhile, β-galactosidase, alkaline phosphatase and conductivity were used to test the damaged effects of fermentation broth on E. coli cell membrane and cell wall. Finally, the effects of protein synthesis of E. coli were detected by coomassie blue method. [Results] The results showed that MIC of JY25 fermentation broth to E. coli was 7 g/L, MBC was 14 g/L, and the fermentation broth with MIC concentration could delay the logarithmic growth of E. coli up to 12 h, with severe deformity and damage of cell morphology. With the extension of treating time, both the β-galactosidase activity and conductivity increased, and the protein synthesis was abnormal. The alkaline phosphatase did not be detected in the broth. [Conclusion] JY25 endophytic fungi from Gynostemma pentaphyllum primarily inhibited the growth of E. coli by damaging the cell membranes and impacting protein synthesis.