The ADP-ribosylation factor 6 (Arf6), as the only member of the Arf family III protein, has been extensively studied for its diverse biological functions in animals. Previously, the Arf6 protein in was found to be crucial for endocytosis and polarity establishment during asexual development. However, its role remains unclear in . Here, we identified and characterized in using a reverse genetic approach. Deletion of impaired hyphal growth and development and produced more branches. Interestingly, knockout of resulted in an augmented tolerance of towards oxidative stress, and increased its sensitivity towards osmotic stress, indicative of the different roles of in various stress responses. Simultaneously, deletion led to an elevation in melanin accumulation. Moreover, the appressorium formation was severely impaired, and fungal virulence to host plants was significantly reduced. Overall, our findings demonstrate the essential role of in hyphal development, stress responses, appressorium formation, and fungal virulence to host plants.