Rubisco activase (Rca) is an essential photosynthetic enzyme that removes inhibitors from the catalytic sites of the carboxylating enzyme Rubisco. In wheat, Rca is composed of one longer 46-kDa α-isoform and two shorter 42-kDa β-isoforms encoded by the genes TaRca1 and TaRca2. TaRca1 produces a single transcript from which a short 1β-isoform is expressed, whereas two alternative transcripts are generated from TaRca2 directing expression of either a long 2α-isoform or a short 2β-isoform. The 2β isoform is similar but not identical to 1β. Here, virus-induced gene silencing (VIGS) was used to silence the different TaRca transcripts. Abundance of the transcripts and the respective protein isoforms was then evaluated in the VIGS-treated and control plants. Remarkably, treatment with the construct specifically targeting TaRca1 efficiently decreased expression not only of TaRca1 but also of the two alternative TaRca2 transcripts. Similarly, specific targeting of the TaRca2 transcript encoding a long isoform TaRca2α resulted in silencing of both TaRca2 alternative transcripts. The corresponding protein isoforms decreased in abundance. These findings indicate concomitant down-regulation of TaRca1 and TaRca2 at both transcript and protein levels and may impact the feasibility of altering the relative abundance of Rca isoforms in wheat.