The mammalian STE20-like kinase-1 (MST1), a multifunctional serine–threonine kinase in mammalian cells, has been recently implicated in the mediation of oxidative stress-induced signaling processes that lead to cell death. However, the molecular mechanism by which oxidative stress induces the stimulation of MST1 remains unclear. In this study, we found that thioredoxin-1 was physically associated with MST1 in intact cells and that this interaction was abolished by H₂O₂. Thioredoxin-1, by binding to the SARAH domain of MST1, inhibited the homodimerization and autophosphorylation of MST1, thereby preventing its activation. Furthermore, TNF-α prevented the physical interaction between thioredoxin-1 and MST1 and promoted the homodimerization and activation of MST1. The effect of TNF-α on MST1 activation was reversed by the reducing agent N-acetyl-l-cysteine. Taken together, our results suggest that thioredoxin-1 functions as a molecular switch to turn off the oxidative stress-induced activation of MST1.