• Vacuolar (H⁺)-PPases (VPs), are key regulators of active proton (H⁺) transport across membranes using the energy generated from PPi hydrolysis. The VPs also play vital roles in plant responses to various abiotic stresses. Their functions in plant responses to pathogen infections are unknown. • Here, we show that TaVP, a VP of wheat (Triticum aestivum) is important for wheat resistance to Chinese wheat mosaic virus (CWMV) infection. Furthermore, overexpression of TaVP in plants induces the activity of PPi hydrolysis, leading to plants cell death. • A virus-derived small interfering RNA (vsiRNA-20) generated from CWMV RNA1 can regulate the mRNA accumulation of TaVP in wheat. The accumulation of vsiRNA-20 can suppress cell death induced by TaVP in a dosage-dependent manner. Moreover, we show that the accumulation of vsiRNA-20 can affect PPi hydrolysis and the concentration of H⁺ in CWMV-infected wheat cells to create a more favorable cellular environment for CWMV replication. • We propose that vsiRNA-20 regulates TaVP expression to prevent cell death and to maintain a weak alkaline environment in cytoplasm to enhance CWMV infection in wheat. This finding may be used as a novel strategy to minimize virus pathogenicity and to develop new antiviral stratagems.