Aims: Mitochondrial thioredoxin (Trx) is critical for defense against oxidative stress-induced cell apoptosis. To date, mitochondrial thioredoxin reductase (TrxR) is the only known enzyme catalyzing Trx2 reduction in mitochondria. However, TrxR is sensitive to inactivation by exo/endogenous electrophiles, for example, 4-hydroxynonenal (HNE). In this study, we characterized the mitochondrial glutaredoxin 2 (Grx2) system as a backup for the mitochondrial TrxR. Meanwhile, as Grx2 is also present in the cytosol/nucleus of certain cancer cell lines, the reducing activity of Grx2 on Trx1 was also tested. Results: Glutathione alone could reduce oxidized Trx2, and the presence of physiological concentrations of Grx2 markedly increased the reaction rate. HeLa cells with Grx2 overexpression (particularly in the mitochondria) exhibited higher viabilities than the wild-type cells after treatment with TrxR inhibitors (Auranofin or HNE), whereas knockdown of Grx2 sensitized the cells to TrxR inhibitors. Accordingly, Grx2 overexpression in the mitochondria had protected Trx2 from oxidation by HNE treatment, whereas Grx2 knockdown had sensitized Trx2 to oxidation. On the other hand, Grx2 reduced Trx1 with similar activities as that of Trx2. Overexpression of Grx2 in the cytosol had protected Trx1 from oxidation, indicating a supportive role of Grx2 in the cytosolic redox balance of cancer cells. Innovation: This work explores the reductase activity of Grx2 on Trx2/1, and demonstrates the physiological importance of the activity by using in vivo redox western blot assays. Conclusion: Grx2 system could help to keep Trx2/1 reduced during an oxidative stress, thereby contributing to the anti-apoptotic signaling. Antioxid. Redox Signal. 21, 669-681.