Murine protein serine–threonine kinase 38 (MPK38) is a member of the AMP‐activated protein kinase-related serine/threonine kinase family. The factors that regulate MPK38 activity and function are not yet elucidated. Here, thioredoxin (Trx) was shown to be a negative regulator of MPK38. The redox-dependent association of MPK38 and Trx was mediated through the C‐terminal domain of MPK38. Single and double amino acid substitution mutagenesis of MPK38 (C286S, C339S, C377S, and C339S/C377S) and Trx (C32S, C35S, and C32S/C35S) demonstrated that Cys339 and Cys377 of MPK38 and Cys32 and Cys35 of Trx are required for MPK38–Trx complex formation. MPK38 directly interacted with and phosphorylated Trx at Thr76. Expression of wild‐type Trx, but not the Trx mutants C32S/C35S and T76A, inhibited MPK38‐induced ASK1, TGF‐β, and p53 function by destabilizing MPK38. The E3 ubiquitin–protein ligase Mdm2 played a critical role in the regulation of MPK38 stability by Trx. Treatment of cells with 1-chloro-2,4-dinitrobenzene, a specific inhibitor of Trx reductase, decreased MPK38–Trx complex formation and subsequently increased MPK38 stability and activity, indicating that Trx negatively regulates MPK38 activity in vivo. Finally, we used ASK1-, Smad3-, and p53-null mouse embryonic fibroblasts to demonstrate that ASK1, Smad3, and p53 play important roles in the activity and function of MPK38, suggesting a functional link between MPK38 and ASK1, TGF-β, and p53 signaling pathways. These results indicate that Trx functions as a physiological inhibitor of MPK38, which plays an important role in inducing ASK1-, TGF‐β-, and p53-mediated activity.