Small ubiquitin-like modifier-1 (SUMO-1) conjugating enzyme 9 (Ubc9) conjugates SUMO-1 to target proteins and modulates cellular processes such as signal transduction, transcription regulation, and cell growth regulation. We demonstrated here that the nucleocapsid protein (NP) of Hantaan virus (HTNV) was associated with Ubc9 and SUMO-1 in vivo. Analysis of the interaction between the truncated NPs and Ubc9 revealed that the amino acid residues at the positions between 101 and 238 in the NP were responsible for the interaction. Furthermore, a consensus binding motif of Ubc9 and SUMO-1, MKAE, within this region, especially the second amino acid of the motif, K residue, was crucial for the interaction, and the interaction was essential for the NP to be localized in the perinuclear region. These results indicate that the assembly of the HTNV-NP is regulated by the interaction between the NP and Ubc9. This is the first report to demonstrate the interaction of Ubc9 with a structural protein of negative-strand RNA viruses.