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Summary Although it is well known that miRNAs play crucial roles in multiple biological processes, there is currently no evidence indicating that milRNAs from Fusarium oxysporum f. sp. lycopersici (Fol) interfere with tomato resistance during infection.Here, using sRNA‐seq, we demonstrate that Fol‐milR1, a trans‐kingdom small RNA, is exported into tomato cells after infection.The knockout strain ∆Fol‐milR1 displays attenuated pathogenicity to the susceptible tomato cultivar ‘Moneymaker’. On the other hand, Fol‐milR1 overexpression strains exhibit enhanced virulence against the resistant cultivar ‘Motelle’. Several tomato mRNAs are predicted targets of Fol‐milR1. Among these genes, Solyc06g007430 (encoding the CBL‐interacting protein kinase, SlyFRG4) is regulated at the posttranscriptional level by Fol‐milR1. Furthermore, SlyFRG4 loss‐of‐function alleles created using CRISPR/Cas9 in tomato (‘Motelle’) exhibit enhanced disease susceptibility to Fol, further supporting the idea that SlyFRG4 is essential for tomato wilt disease resistance. Notably, our results using immunoprecipitation with specific antiserum suggest that Fol‐milR1 interferes with the host immunity machinery by binding to tomato ARGONAUTE 4a (SlyAGO4a). Furthermore, virus‐induced gene silenced (VIGS) knock‐down SlyAGO4a plants exhibit reduced susceptibility to Fol.Together, our findings support a model in which Fol‐milR1 is an sRNA fungal effector that suppresses host immunity by silencing a disease resistance gene, thus providing a novel virulence strategy to achieve infection., See also the Commentary on this article by Thieron et al., 232: 464–467.