Flavodoxins play central roles in the electron transfer involving various biological processes in microorganisms. The mioC gene of Escherichia coli encodes a 16 kDa flavodoxin and locates next to the chromosomal replication initiation origin (oriC). Extensive researches have been carried out to investigate the relationship between mioC transcription and replication initiation. Recently, the MioC protein was proposed to be essential for the biotin synthase activity in vitro. Nevertheless, the exact role of MioC in biotin synthesis and its physiological function in vivo remain elusive. In order to understand the molecular basis of the biological functions of MioC and the cofactor-binding mechanisms of flavodoxins, we have determined the solution structures of both the apo- and holo-forms of E. coli MioC protein at high resolution by nuclear magnetic resonance spectroscopy. The overall structures of both forms consist of an a/ß sandwich, which highly resembles the classical flavodoxin fold. However, significant diversities are observed between the two forms, especially the stabilization of the FMN-binding loops and the notable extension of secondary structures upon FMN binding. Structural comparison reveals fewer negative charged and aromatic residues near the FMN-binding site of MioC, as compared to that of flavodoxin 1 from E. coli, which may affect both the redox potentials and the redox partner interactions. Furthermore, the backbone dynamics studies reveal the conformational flexibility at different timescales for both apo- and holo-forms of MioC, which may play important roles for cofactor binding and electron transfer.