Agrobacterium tumefaciens causes crown gall disease by transferring its DNA into host plants. Although Agrobacterium can be popularly used for genetic engineering, above-ground insect infestation in Agrobacterium gall formation has not been investigated. Nicotiana benthamiana leaves were exposed to a sucking insect whitefly infestation and a chemical trigger, benzothiadiazole (BTH), for 7 days, and these exposed plants were inoculated with Agrobacterium. We evaluated how whitefly infestation manipulated gall disease by Agrobacterium in planta and in vitro. Insect whitefly infested plants exhibited at least a 2-fold reduction in gall formation on both stem and crown root. Silencing isochorismate synthase 1 (ICS1), required for salicylic acid synthesis, compromised gall formation, indicating an involvement of salicylic acid in whitefly-derived plant defense against Agrobacterium. Endogenous salicylic acid content was augmented in whitefly-infested plants by Agrobacterium inoculation. However, infestation with whitefly did not alter Agrobacterium root colonization but reduced expression levels of genes involved in Agrobacterium virulence and transformation efficiency. Above-ground whitefly infestation therefore elicits systemic responses throughout the plant. Our findings provide new insights into insect-mediated leaf-root intra-communication and a framework to understand a general principle in multitrophic interactions in nature.