Thioredoxin (Trx) and thioredoxin reductase 1 (TR1) are among the major redox regulators in mammalian cells and have a wide variety of roles, including removal of intracellular reactive oxygen species (ROS) and prevention of cell death. Tumor necrosis factor-α (TNF-α) induces cancer cell death. Although ROS have been proposed to participate in this process, the role of the thioredoxin system in TNF-α stimulated cell death remains unclear. We investigated the possibility that the thioredoxin system protects against TNF-α-induced cancer cell death by examining whether TR1/Trx1 status controls TNF-α-induced apoptosis in EMT6 murine breast cancer cells. TR1-deficient cells were more sensitive to TNF-α than control cells. Increased sensitivity to TNF-α was most pronounced in Trx1-deficient cells. TNF-α-induced nuclear localization of phosphorylated ERK 1/2 (p-ERK 1/2) correlated with increased apoptosis in TR1- and Trx1-deficient cells, suggesting a pro-apoptotic role for nuclear p-ERK 1/2 in TNF-α-induced apoptosis. In addition, phosphoinositide 3-kinase (PI3K) inhibition dramatically reduced TNF-α-stimulated apoptosis and nuclear localization of p-ERK 1/2. In contrast, inhibition of ROS, MEK, JNK, or p38 did not significantly alter p-ERK 1/2 localization or apoptosis in TR1- and Trx1-deficient cells compared to control cells. Further, NF-κB p65 localization was not changed in TR1- and Trx1-deficient cells in response to TNF-α relative to control cells. Our data suggest that the thioredoxin system plays a critical role in protecting against TNF-α-induced apoptosis by regulating the levels of nuclear p-ERK 1/2 in a PI3K-dependent manner.