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 infiltration-mediated RNA-silencing assays, we demonstrated that GLRaV-1 p24 protein (p24) acts as an RNA-silencing suppressor (RSS), inhibiting local and systemic RNA silencing. Electrophoretic mobility shift assays showed that p24 binds double-stranded 21-nucleotide small interfering RNA (siRNA), and that siRNA binding is required but not sufficient for its RSS activity. p24 localizes in the nucleus and can self-interact through its amino acid 10 to 210 region. Dimerization is needed for p24 interaction with importin α1 before moving to the nucleus, but is not required for its siRNA binding and RSS activity. Expression of p24 from a binary pGD vector or potato virus X-based vector elicited a strong hypersensitive response in species, indicating that p24 may be a factor in pathogenesis. Furthermore, p24 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. These results contribute to our understanding of the molecular mechanisms underlying GLRaV-1 infection.