Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the most important viruses of grapevine but, despite this, there remain several gaps in our understanding of its biology. Because of its narrow host range - limited to Vitis species - and because the virus is restricted to the phloem, most GLRaV-3 research has concentrated on epidemiology and the development of detection assays. The recent discovery that GLRaV-3 can infect Nicotiana benthamiana, a plant model organism, makes new opportunities available for research in this field. We used RNA-seq to compare both V. vinifera and P1/HC-Pro N. benthamiana host responses to GLRaV-3 infection. Our analysis revealed that the majority of DEGs observed between the two hosts were unique although responses between the two hosts also showed several shared gene expression results. When comparing gene expression patterns that were shared between the two hosts, we observed the downregulation of genes associated with stress chaperones, and the induction of gene families involved in primary plant physiological processes. This is the first analysis of gene expression profiles beyond Vitis to mealybug-transmitted GLRaV-3 and demonstrates that N. benthamiana could serve as a useful tool for future studies of GLRaV-3-host interactions.