Abstract: The RNAi vector of the target gene was transfected into cucumber (Cucumis sativus) cotyledons and cotyledon nodes by Agrobacterium tumefaciens-mediated transfection. A total of 99 regeneration plants were hygromycin-resistant. PCR and Southern-blot analysis revealed that target gene was integrated into the genome of 10 regenerated plants. The transformation efficiency in the cucumber cotyledon and cotyledon node was significantly increased from 4.1% to 13.9% and 3.1% to 6.9%, respectively, when using different hygromycin concentrations in the selection cultivation. We have optimized the genetic transformation system with an RNA interference vector in cucumber and successfully obtained transgenic cucumber plants.