Glutathione is a metabolite that plays an important role in plant response to biotic stress through its ability to remove reactive oxygen species, thereby limiting the degree of potential oxidative damage. It can couple changes in the intracellular redox state to the development, especially the defense responses, of plants. Several studies have focused on measuring glutathione levels in virus infected plants, but have not provided complete information. Therefore, we analyzed, for the first time, the content of glutathione as well as its ultrastructural distribution related to susceptible and hypersensitive potato–Potato virus Y NTN (PVYNTN) interaction, with an aim of providing new insight into interactive responses to PVYNTN stress. Our findings reported that the inoculation of PVYNTN caused a dynamic increase in the content of glutathione, not only in resistance but also in susceptible reaction, especially at the first steps of plant–virus interaction. Moreover, the increase in hypersensitive response was much more dynamic, and accompanied by a significant reduction in the content of PVYNTN. By contrast, in susceptible potato Irys, the content of glutathione decreased between 7 and 21 days after virus inoculation, which led to a significant increase in PVYNTN concentration. Additionally, our findings clearly indicated the steady induction of two selected potato glutathione S-transferase StGSTF1 and StGSTF2 genes after PVYNTN inoculation, regardless of the interaction type. However, the relative expression level of StGSTF1 did not significantly differ between resistant and susceptible plants, whereas the relative expression levels of StGSTF2 differed between susceptible and resistant reactions. Therefore, we proposed that StGSTF2 can act as a marker of the type of response to PVYNTN. Our observations indicated that glutathione is an important component of signaling as well as the regulatory network in the PVYNTN–potato pathosystem. In resistance responses to PVYNTN, this metabolite activates plant defenses by reducing potential damage to the host plant cell, causing a reduction in virus concentration, while it can also be involved in the development of PVYNTN elicited symptoms, as well as limiting oxidative stress, leading to systemic infection in susceptible potato plants.