Background: In our previous study, the novel anti-Verticilliwn dahliae protein TUBP1 was isolated from Bacillus azarquiensis, and the anti-V. dahliae effect was investigated. In the present study, the membrane-active action 01 the TUBP1 protein on V. dahliae was evaluated. Result Experiments were conducted to examine plasma membrane lesions and membrane depolarization, which were dyed using propidium iodide (PI) and 3,3'-dipropylthiacarbocyanine [DiSC3(5)], respectively. The result: showed perturbation of the plasma membrane of the spores and the hyphae of V. dahliae when they were treated with the TUBPI protein. Atomic absorption spectrometry (AAS) was used to analyze the K+ concentration. The results showed that through releasing K+, the TUBP1 protein induced membrane damage and depolarization FITC-labeled TUBP1 protein influx, calcein leakage from liposomes, and pore size of the spores and hyphae of V. dahliae experiments were performed with FITC-labeled TUBP1 protein, calcein-encapsulating large unilamellai vesicles (LUV), and edextran (FD) loaded LUVs, respectively. The results demonstrated that the negative effect 01 the TUBP1 protein on spores and hyphae plasma membranes of V. dahliae was apparent in the formation of pore: with radii from 1.4 to 2.3 nm. Finally, further morphological changes in V. dahliae spores and hyphae induced 133 the TUBP1 protein were observed in the results of flow cytometry and scanning electron microscopy (SEM). Conclusion: These results showed that in three-dimensional flow cytometric contour plots, cell sizes were re duced by the pore-forming action of TUBP1 protein.