Post-translational modifications of host or viral proteins are key strategies exploited by viruses to support virus replication and counteract host immune response. SUMOylation is a post-translational modification process mediated by a family of ubiquitin-like proteins called small ubiquitin-like modifier (SUMO) proteins. Multiple sequence alignment of 78 representative flaviviruses showed that most (72/78, 92.3%) have a putative SUMO-interacting motif (SIM) at their non-structural 5 (NS5) protein’s N-terminal domain. The putative SIM was highly conserved among 414 pre-epidemic and epidemic Zika virus (ZIKV) strains, with all of them having a putative SIM core amino acid sequence of VIDL (327/414, 79.0%) or VVDL (87/414, 21.0%). Molecular docking predicted that the hydrophobic SIM core residues bind to the β2 strand of the SUMO-1 protein, and the acidic residues flanking the core strengthen the binding through interactions with the basic surface of the SUMO protein. The SUMO inhibitor 2-D08 significantly reduced replication of flaviviruses and protected cells against ZIKV-induced cytopathic effects in vitro. A SIM-mutated ZIKV NS5 failed to efficiently suppress type I interferon signaling. Overall, these findings may suggest SUMO modification of the viral NS5 protein to be an evolutionarily conserved post-translational modification process among flaviviruses to enhance virus replication and suppress host antiviral response.