In this study, we determined the crystal structure of N-terminal importin-beta-binding domain (IBB)-truncated human importin-alpha1 (DeltaIBB-h-importin-alpha1) at 2.63 A resolution. The crystal structure of DeltaIBB-h-importin-alpha1 reveals a novel closed homodimer. The homodimer exists in an autoinhibited state in which both the major and minor nuclear localization signal (NLS) binding sites are completely buried in the homodimerization interface, an arrangement that restricts NLS binding. Analytical ultracentrifugation studies revealed that DeltaIBB-h-importin-alpha1 is in equilibrium between monomers and dimers and that NLS peptides shifted the equilibrium toward the monomer side. This finding suggests that the NLS binding sites are also involved in the dimer interface in solution. These results show that when the IBB domain dissociates from the internal NLS binding sites, e.g., by binding to importin-beta, homodimerization possibly occurs as an autoinhibition state.