Cross-kingdom RNA interference (ckRNAi) is a mechanism of interspecies communication where small RNAs (sRNAs) are transported from one organism to another, where they silence target genes in trans1-9. ckRNAi is well established in pathosystems1-9 and has also been observed in a small number of mutualistic interactions10,11. Fungal and oomycete pathogens, as well as symbiotic bacterium, send sRNAs into host cells to hijack host RNAi machinery by loading into host AGO proteins in order to silence host target genes. Previous research from our group has suggested the possibility of ckRNAi occurring in Arbuscular Mycorrhizal Symbiosis (AMS)12, the most widespread and ancient symbiosis on Earth13. Here, we show that a specific sRNA from the AM fungus Rhizophagus irregularis (Rir2216) is loaded into an AGO1 silencing complex from the host plant Medicago truncatula, leading to cleavage of a host target transcript encoding for the MtWRKY69 transcription factor. MtWRKY69 is downregulated in arbusculated cells in mycorrhizal roots and MtWRKY69 overexpression led to a reduced AM colonization level, indicating that its silencing is relevant for AMS. Thus, we present the first experimental evidence of the molecular mechanism of fungus to plant ckRNAi in AMS.