The properties of CO-inhibited Azotobacter vinelandii (Av) Mo-nitrogenase (N2ase) have been examined by the combined application of nuclear resonance vibrational spectroscopy (NRVS), extended X-ray absorption fine structure (EXAFS), and density functional theory (DFT). Dramatic changes in the NRVS are seen under high-CO conditions, especially in a 188 cm–1 mode associated with symmetric breathing of the central cage of the FeMo-cofactor. Similar changes are reproduced with the α-H195Q N2ase variant. In the frequency region above 450 cm–1, additional features are seen that are assigned to Fe-CO bending and stretching modes (confirmed by 13CO isotope shifts). The EXAFS for wild-type N2ase shows evidence for a significant cluster distortion under high-CO conditions, most dramatically in the splitting of the interaction between Mo and the shell of Fe atoms originally at 5.08 Å in the resting enzyme. A DFT model with both a terminal −CO and a partially reduced −CHO ligand bound to adjacent Fe sites is consistent with both earlier FT-IR experiments, and the present EXAFS and NRVS observations for the wild-type enzyme. Another DFT model with two terminal CO ligands on the adjacent Fe atoms yields Fe-CO bands consistent with the α-H195Q variant NRVS. The calculations also shed light on the vibrational “shake” modes of the interstitial atom inside the central cage, and their interaction with the Fe-CO modes. Implications for the CO and N2 reactivity of N2ase are discussed.