The transcription factor NF-κB, a central regulator of immunity, is subject to regulation by redox changes. We now report that cysteine-179 of the inhibitory κB kinase (IKK) β-subunit of the IKK signalosome is a central target for oxidative inactivation by means of S-glutathionylation. S-glutathionylation of IKK-β Cys-179 is reversed by glutaredoxin (GRX), which restores kinase activity. Conversely, GRX1 knockdown sensitizes cells to oxidative inactivation of IKK-β and dampens TNF-α-induced IKK and NF-κB activation. Primary tracheal epithelial cells from Glrx1-deficient mice display reduced NF-κB 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-κB pathway.