Glutathione could be a crucial cell molecule during plant–pathogen interplay response via its role in the direct control level of ROS. The proper regulation of ROS creates a line between susceptibility (with high damage to cell components) and host resistance in some plant–pathogen interactions. Therefore, we analyzed the role of glutathione forms (GSH/GSSG), as well as their cellular localization, in compatible and incompatible (HR reaction) potato cultivars infected with Potato virus Y ^NTN (PVY^NTN) during interaction in the symplast and apoplast with the use of microscopic and HPLC methods. Only the resistant potato plants characterized systemically increased glutathione production in both cell compartments, accompanied by a significant reduction in virus level. The susceptible potato plants were only able to induce glutathione at an early stage of infection; after that, glutathione levels were depleted, and virus concentration highly increased. This fact directly indicates that proper glutathione synthesis and concertation in the symplast and apoplast plays a role not only as anti-ROS protection but also as an antiviral factor in the infection of PVY^NTN. Whereas the apoplastic overpresence of GSSG and reduction in glutathione that occurred in the symplast are clear pro-viral factors for PVY^NTN. Therefore, supporting plant supplementation with glutathione could be a factor that changes/stimulates more resistance reactions to PVY^NTN infection.