The V2 protein of Tomato yellow leaf curl geminivirus (TYLCV), encoded by the v2 gene, functions as an RNA-silencing suppressor that counteracts the innate immune response of the host plant. In this study, we analyzed the effect of small interfering double-stranded RNAs (siRNAs), derived from an intron-hairpin RNA (ihpRNA) construct and targeting the v2 gene product, on its expression in planta. Transient assays involving agroinfiltration of the V2 protein-silencing construct followed by infiltration of a fused GFP-V2 (green fluorescent -V2 protein) gene showed down-regulation of GFP expression in Nicotiana benthamiana indicating silencing of the fused v2-GFP RNA. Transgenic tobacco plants expressing the siRNA targeted against the TYLCV V2 gene showed inactivation of the V2 suppression of RNA silencing activity, thereby enabling silencing of agroinfiltrated GFP gene. The present study demonstrates that siRNA targeted against the v2 gene product of TYLCV can lead to down regulation of its expression and can be used to further study TYLCV V2 protein functions in the host cell.