Grapevine leafroll-associated virus 1 (GLRaV-1) is a major pathogen associated with grapevine leafroll disease. However, the molecular mechanisms underlying GLRaV-1 interactions with plant cells are unclear. Using Agrobacterium infiltration-mediated RNA-silencing assays, we demonstrated that GLRaV-1 p24 protein (p24(G1)) acts as an RNA-silencing suppressor (RSS), inhibiting local and systemic RNA silencing. Electrophoretic mobility shift assays showed that p24(G1) binds double-stranded 21-nucleotide small interfering RNA (siRNA), and that siRNA binding is required but not sufficient for its RSS activity. p24(G1) localizes in the nucleus and can self-interact through its amino acid 10 to 210 region. Dimerization is needed for p24(G1) interaction with importin alpha 1 before moving to the nucleus, but is not required for its siRNA binding and RSS activity. Expression of p24(G1) from a binary pGD vector or potato virus X-based vector elicited a strong hypersensitive response in Nicotiana species, indicating that p24(G1) may be a factor in pathogenesis. Furthermore, p24(G1) function in pathogenesis required its RSS activity, dimerization and nuclear localization. In addition, the region of amino acids 122-139 played a crucial role in the nuclear import, siRNA binding, silencing suppression and pathogenic activity of p24(G1). These results contribute to our understanding of the molecular mechanisms underlying GLRaV-1 infection.