The redox-sensitive OxyR protein activates the transcription of antioxidant defense genes in response to oxidative stress and represses its own expression under both oxidizing and reducing conditions. Previous studies showed that OxyR-binding sites are unusually long with limited sequence similarity. Here, we report that oxidized OxyR recognizes a motif comprised of four ATAGnt elements spaced at 10 bp intervals and contacts these elements in four adjacent major grooves on one face of the DNA helix. In contrast, reduced OxyR contacts two pairs of adjacent major grooves separated by one helical turn. The two modes of binding are essential for OxyR to function as both an activator and a repressor in vivo. We propose that specific DNA recognition by an OxyR tetramer is achieved with four contacts of intermediate affinity allowing OxyR to reposition its DNA contacts and target alternate sets of promoters as the cellular redox state is altered.