The strong interaction between streptavidin (SA) and biotin is widely utilized in biotechnological applications. A SA variant, monovalent SA, was developed with a single and high affinity biotin-binding site within the intact tetramer. However, its structural characterization remains undetermined. Here, we seek to determine the crystal structure of monovalent SA at 1.7-Å resolution. We show that, in contrast to its 'close-state' in the only wild-type subunit, the L3,4 loops of three Dead SA subunits are free from crystal packing and remain in an 'open state', stabilized by a consistent H-bonding network involving S52. This H-bonding network also applies to the previously reported open state of the wild-type apo-SA. These results suggest that specific substitutions (N23A/S27D/S45A) at biotin-binding sites stabilize the open state of SA L3,4 loop, thereby further reducing biotin-binding affinity. The general features of the 'open state' SA among different SA variants may facilitate its rational design. The structural information of monovalent SA will be valuable for its applications across a wide range of biotechnological areas.