Mutations of the DNA methyltransferase 3B (DNMT3B) gene have been detected in patients with immunodeficiency, centromere instability, and facial anomalies (ICF) syndrome. Most of these mutations are clustered in its catalytic domain and thus lead to defective DNA methylation. Nevertheless, the S270P mutation in the N-terminal PWWP (Pro-Trp-Trp-Pro) domain of the DNMT3B gene has prompted questions as to how this mutation contributes to the development of ICF syndrome. In this study, we found that wild-type DNMT3B is SUMOylated through covalent modification, whereas the S270P mutant interacts with SUMO-1 via non-covalent interaction. The S270P mutation results in diffuse nucleus localization. Moreover, the S270P mutant fails to interact with PIAS1, a small ubiquitin-related modifier (SUMO) E3 ligase, and causes the constitutive activation of nuclear factor-kappa B, which induces the expression of interleukin 8. Collectively, our data demonstrate that the S270P mutation affects DNMT3B functions via specific, non-covalent interaction with SUMO-1.