This study identifies glutathione S-transferase Pi (GSTp) as a novel endogenous inhibitor of Jun N-terminal kinase (JNK) signaling. GSTp, a member of the glutathione S-transferase family, was purified and characterized as a JNK-associated protein that inhibits JNK activity. The monomeric form of GSTp is responsible for JNK inhibition, while oligomerization of GSTp upon stress (e.g., UV irradiation or H₂O₂ treatment) leads to dissociation of the GSTp-JNK complex, thereby releasing JNK activity. GSTp inhibits JNK activity in a dose-dependent manner, and its inhibitory effect is reversed by specific GSTp inhibitors or a GSTp-derived peptide. GSTp expression reduces JNK phosphorylation, c-Jun ubiquitination, and c-Jun-mediated transcription, while its absence leads to increased JNK activity. GSTp inhibition of JNK is independent of the MEKK1-MKK4 signaling module. In GSTp-null mice, JNK activity is elevated, and forced expression of GSTp reduces this activity. GSTp's inhibitory effect on JNK is mediated by its monomeric form, which is disrupted by stress-induced dimerization. GSTp also influences JNK activity through its interaction with the Jun–JNK complex, and its inhibitory effect is not dependent on GSTp's enzymatic activity. The study highlights the role of GSTp in regulating JNK signaling through its redox-sensitive conformational changes, providing new insights into the regulation of stress kinases.This study identifies glutathione S-transferase Pi (GSTp) as a novel endogenous inhibitor of Jun N-terminal kinase (JNK) signaling. GSTp, a member of the glutathione S-transferase family, was purified and characterized as a JNK-associated protein that inhibits JNK activity. The monomeric form of GSTp is responsible for JNK inhibition, while oligomerization of GSTp upon stress (e.g., UV irradiation or H₂O₂ treatment) leads to dissociation of the GSTp-JNK complex, thereby releasing JNK activity. GSTp inhibits JNK activity in a dose-dependent manner, and its inhibitory effect is reversed by specific GSTp inhibitors or a GSTp-derived peptide. GSTp expression reduces JNK phosphorylation, c-Jun ubiquitination, and c-Jun-mediated transcription, while its absence leads to increased JNK activity. GSTp inhibition of JNK is independent of the MEKK1-MKK4 signaling module. In GSTp-null mice, JNK activity is elevated, and forced expression of GSTp reduces this activity. GSTp's inhibitory effect on JNK is mediated by its monomeric form, which is disrupted by stress-induced dimerization. GSTp also influences JNK activity through its interaction with the Jun–JNK complex, and its inhibitory effect is not dependent on GSTp's enzymatic activity. The study highlights the role of GSTp in regulating JNK signaling through its redox-sensitive conformational changes, providing new insights into the regulation of stress kinases.