RNAi technologies have been exploited to control viruses, pests, oomycetes and fungal phytopathogens that cause disasters in host plants, including many agronomically significant crops. However, it is unclear what process mediates RNA uptake by fungi. Here, the authors utilized live-cell imaging technology combined with molecular biology experiments to demonstrate that exogenous RNA is indiscriminately absorbed by Verticillium dahliae, the notorious plant pathogenic fungus. The uptake of exogenous RNA by fungal cells is predominantly mediated through endocytosis. This study not only provides a new theoretical foundation for applying trans-kingdom RNA interference technology in crop protection but also lays the groundwork for research and applications of exogenous RNA in plant-fungi interaction systems.