This study identifies glutathione S-transferase Pi (GSTp) as a JNK inhibitor in non-stressed cells. GSTp, which is present in low basal levels, associates with the Jun–JNK complex and inhibits JNK activity in a dose-dependent manner. UV irradiation or H2O2 treatment causes GSTp oligomerization and dissociation from the Jun–JNK complex, indicating that monomeric GSTp is responsible for JNK inhibition. GSTp expression in mouse embryo fibroblasts from GSTp-null mice reduces basal JNK activity and increases c-Jun ubiquitination and transcriptional activity. GSTp inhibition of JNK is independent of MKK4 phosphorylation and MEKK1-MKK4 signaling modules. The monomeric form of GSTp mediates JNK inhibition, and GSTp-induced JNK activation can be blocked by GSTp inhibitors and GSTp-derived peptides. These findings provide new insights into the regulation of stress kinases by GSTp.This study identifies glutathione S-transferase Pi (GSTp) as a JNK inhibitor in non-stressed cells. GSTp, which is present in low basal levels, associates with the Jun–JNK complex and inhibits JNK activity in a dose-dependent manner. UV irradiation or H2O2 treatment causes GSTp oligomerization and dissociation from the Jun–JNK complex, indicating that monomeric GSTp is responsible for JNK inhibition. GSTp expression in mouse embryo fibroblasts from GSTp-null mice reduces basal JNK activity and increases c-Jun ubiquitination and transcriptional activity. GSTp inhibition of JNK is independent of MKK4 phosphorylation and MEKK1-MKK4 signaling modules. The monomeric form of GSTp mediates JNK inhibition, and GSTp-induced JNK activation can be blocked by GSTp inhibitors and GSTp-derived peptides. These findings provide new insights into the regulation of stress kinases by GSTp.