Powdery mildew disease, caused by Blumeria graminis f. sp. hordei (Bgh), which belongs to the order Erysiphales, is a major crop disease. The general control nondepressible-2 (GCN2) gene of barley was previously found to be overexpressed during the powdery mildew resistance response. Recently, Arabidopsis thaliana GCN2 (AtGCN2) was shown to be involved in disease resistance against biotrophic and necrotrophic pathogens. In order to understand the function of Hordeum vulgare GCN2 (HvGCN2) in the barley powdery mildew resistance response, this gene was silenced by barley stripe mosaic virus (BSMV), mediated by virus-induced gene silencing (VIGS). This is the first study showing the potential importance of HvGCN2 in powdery mildew disease of barley. Based on our observations, when HvGCN2 was silenced on average by 53.5%, Bgh development was increased by 18.7 to 32.1%, which was determined by primary, secondary and longest hyphae measurements. The number of germinated spores also increased 2.8-fold in HvGCN2 silenced plants compared to control plants (BSMV:00). On the other hand, under the resistant condition, no difference was observed in HvGCN2-silenced plants compared to non-silenced lines although the gene was found to be overexpressed in incompatible interaction.