The transcription factor NF-kappaB, a central regulator of immunity, is subject to regulation by redox changes. We now report that cysteine-179 of the inhibitory kappaB kinase (IKK) beta-subunit of the IKK signalosome is a central target for oxidative inactivation by means of S-glutathionylation. S-glutathionylation of IKK-beta Cys-179 is reversed by glutaredoxin (GRX), which restores kinase activity. Conversely, GRX1 knockdown sensitizes cells to oxidative inactivation of IKK-beta and dampens TNF-alpha-induced IKK and NF-kappaB activation. Primary tracheal epithelial cells from Glrx1-deficient mice display reduced NF-kappaB DNA binding, RelA nuclear translocation, and MIP-2 (macrophage inflammatory protein 2) and keratinocyte-derived chemokine production in response to LPS. Collectively, these findings demonstrate the physiological relevance of the S-glutathionylation-GRX redox module in controlling the magnitude of activation of the NF-kappaB pathway.