Plasmopara viticola causes grape downy mildew, one of the most notorious diseases of cultivated grapes that damage vineyards worldwide. The pathogen secretes various effector molecules to infect and modulate the host biological processes. In this study, we aimed to evaluate the roles of KPvRxLR27, an arginine-any amino acid-leucine-arginine (RxLR) effector isolated from P. viticola JN-9 from Jeonju (South Korea) with respect to the reported Bcl-2-associated X and inverted formin1in inducing cell death in non-host Nicotiana benthamiana and resistant grape host cultivars via Agrobacterium-mediated transient transformation. We found that, KPvRxLR27 induced programmed cell death in N. benthamiana and rapid hypersensitive response in resistant grape cultivars. Agroinfiltration assay revealed that putative N-glycosylation at the N186 amino acid sequence and nuclear localization signal motifs at the C-terminus were critical for the effector's cell death-inducing activity of KPvRxLR27. Overexpression assay revealed that KPvRxLR27 was abundantly expressed in the plasma membrane and nuclear regions and activated the accumulation of reactive oxygen species in N. benthamiana. Moreover, KPvRxLR27 expression was significantly delayed in the resistant cultivar than in the susceptible cultivar. Our results suggest KPvRxLR27 as a potential avirulence gene recognized by the host receptors to activate the host immune response-associated genes, providing valuable insights to enhance the pathogen resistance of commercial cultivars.