The RADIATION SENSITIVE23 (RAD23) transfers ubiquitylated substrates to proteasome and has a role in protein degradation. In our previous proteomics study, RAD23 protein was found to be over-expressed at 12 hours post inoculation (hpi) in compatible interaction (Pallas-03 inoculated with Blumeria graminis f.sp. hordei (Bgh) race 103); however it was not detectable in incompatible interaction (Pallas-01 inoculated with Bgh-103). Thus, to assess its role in barley powdery mildew resistance, the response to the virulent and avirulent pathogen races was studied on the RAD23 silenced barley seedlings using Barley Stripe Mosaic Virus (BSMV). Control plants (BSMV:00) were compared with the RAD23 silenced plants (BSMV :HvRAD23) after they are inoculated with both Bgh-103 (virulent) and Bgh-95 (avirulent) pathogen races. Hyphal growth levels were analyzed at 3 and 5 days post inoculation (dpi). HvRAD23 was found to be silenced on average 17.3% in three biological replicates. Despite the low silencing level, the change in hyphal growth was statistically significant. The primary, secondary and longest hyphae lengths were found to be decreased 21, 22 and 16%, respectively. This result together with the proteomics data suggested the idea that RAD23 protein is a negative regulator of powdery mildew resistance in barley.