Counteracting plant RNA silencing ensures successful viral infection. The P25 protein encoded by potato virus X (PVX) is a multifunctional protein that acts as a viral RNA silencing suppressor (VSR). In this study, we screened out a potato type I protease inhibitor (PI) in Nicotiana benthamiana (NbPI) that interacts with P25. Silencing of NbPI by tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) promoted the infection of PVX. Overexpression of NbPI in transgenic plants conferred resistance to PVX infection. Moreover, transient expression of NbPI impaired the VSR activity and cell-to-cell movement complementation ability of P25. Further experiments showed that P25 protein degradation was through the combination of autophagy and the ubiquitin-26S proteasome system (UPS), leading to impairment of P25. Taken together, we have identified NbPI as a new host factor that compromises PVX infection by targeting and degrading the VSR P25.