The Anthracis repressor (AntR) is a Mn(II)-activated DNA binding protein that is involved in the regulation of Mn(II) homeostasis in Bacillus anthracis. AntR is structurally and functionally homologous to Mn(II)-activated repressor from Bacillus subtillis (MntR). Our studies on AntR focus on metal-regulated activation of the protein. Line shape analysis of continuous wave electron paramagnetic resonance (EPR) spectra showed that metal binding resulted in a general reduction of backbone dynamics and that there were no further changes in backbone motion upon DNA binding. Double electron-electron resonance (DEER) pulsed EPR spectroscopy was used to measure distances between nitroxide spin labels strategically placed in dimeric AntR. The DEER data were analyzed assuming Gaussian distributions for discrete populations of spins. A structural model for AntR was built from homology to MntR, and the experimentally measured distances were simulated to distinguish between spin label and backbone motions. Together with the computational analysis, the DEER results for apo-AntR indicated relatively narrow conformational distributions for backbone residues at the dimer interface and near the metal binding site. No significant changes were observed on these sites in the presence of metal or DNA. On the other hand, the distribution of the conformers and the distances between the putative DNA binding helices decreased upon metal binding. These results suggest that the DNA binding region of AntR shows large amplitude backbone motions in the absence of metal, which may preclude sequence-specific binding to promoter sites. Metal binding narrows the range of conformations accessible in this region and shortens the mean distance between the DNA binding helices, probably resulting in alignment that optimizes promoter recognition and binding.