Rhizomania is an economically important disease of sugar beet, which is caused by Beet necrotic yellow vein virus (BNYVV). As previously shown, RNA silencing mechanism effectively inhibit the viral propagation in transgenic sugar beet plants. To investigate possible proteomic changes induced by gene insertion and/or RNA silencing mechanism, the root protein profiles of wild type sugar beet genotype 9597, as a control, and transgenic events named 6018-T3:S6-44 (S6) and 219-T3:S3-13.2 (S3) were compared by two-dimensional gel electrophoresis. The accumulation levels of 25 and 24 proteins were differentially regulated in S3 and S6 plants, respectively. The accumulation of 15 spots were increased or decreased more than 2-fold. Additionally, 10 spots repressed or induced in both, while seven spots showed variable results in two events. All the differentially expressed spots were analyzed by MALDI-TOF-TOF mass spectrometry. The functional analysis of differentially accumulated proteins showed that most of them are related to the metabolism and defense/stress response. None of these recognized proteins were allergens or toxic proteins except for a spot identified as phenylcoumaran benzylic ether reductase, Pyrc5, which was decreased in the genetically modified S6 plant. These data are in favor of substantial equivalence of the transgenic plants in comparison to their related wild type cultivar since the proteomic profile of sugar beet root was not remarkably affected by gene transfer and activation RNA silencing mechanism.