Summary Pepino mosaic virus (PepMV) is pandemic in tomato crops, causing important economic losses world‐wide. No PepMV‐resistant varieties have been developed yet. Identification of host factors interacting with PepMV proteins is a promising source of genetic targets to develop PepMV‐resistant varieties. The interaction between the PepMV coat protein (CP) and the tomato glutathione S‐transferase (GST) SlGSTU38 was identified in a yeast two‐hybrid (Y2H) screening and validated by directed Y2H and co‐immunoprecipitation assays. SlGSTU38‐knocked‐out Micro‐Tom plants (gstu38) generated by the CRISPR/Cas9 technology together with live‐cell imaging were used to understand the role of SlGSTU38 during infection. The transcriptomes of healthy and PepMV‐infected wild‐type (WT) and gstu38 plants were profiled by RNA‐seq analysis. SlGSTU38 functions as a PepMV‐specific susceptibility factor in a cell‐autonomous manner and relocalizes to the virus replication complexes during infection. Besides, knocking out SlGSTU38 triggers reactive oxygen species accumulation in leaves and the deregulation of stress‐responsive genes. SlGSTU38 may play a dual role: On the one hand, SlGSTU38 may exert a proviral function depending on its specific interaction with the PepMV CP; and on the other hand, SlGSTU38 may delay PepMV‐infection sensing by participating in the redox intracellular homeostasis in a nonspecific manner.