RNAi technologies have been exploited to control viruses, pests, oomycetes, and fungal phytopathogens that cause disasters in host plants, including many agronomically significant crops. Double-stranded RNA (dsRNA) or small interfering RNA (siRNA) has been applied as a trigger for trans-kingdom RNAi between hosts and fungi. However, it is unclear what process mediates RNA uptake by fungi. In this study, by using live-cell imaging, we determined that exogenously synthesized RNA or small RNA (sRNA) was indiscriminately absorbed into Verticillium dahliae, a notorious pathogenic fungus. Moreover, the application of endocytic inhibitors or deletion of endocytic-related genes reduced RNA uptake efficiency, showing that RNA absorption by fungal cells occurs mainly through endocytosis. In addition, we found that the endocytosed fluorescence-labeled RNAs were partly colocalized with endosome marker genes. Overall, our research concluded that exogenous RNA could be assimilated by V. dahliae through the endocytic pathway. Unraveling this cytological mechanism underlying trans-kingdom RNAi holds significant importance, especially considering the fact that RNAi-based strategies targeting pathogenic fungi are increasingly prevalent in the realm of crop protection.