Coussens et al. utilized the high-throughput screening to identify five small-molecules as inhibitors of the catalytic SET domain and compound 3 exhibited apparent activity in cells. Freitas et al. reported the first inhibitor (4) of NSD2-PWWP1 domain with K d value about 3.4 μM in an SPR assay, which could abrogate histone H3K36me2 binding to the PWWP1 domain in cells with an IC 50 of 17.3 μM. Lately, Dilworth et Since there is only one chemotype of NSD2-PWWP1 inhibitors that showed biological activity, therefore, more diverse chemical scaffolds and potent NSD2-PWWP1 inhibitors are needed to further assess the pharmacological potential of NSD2. Herein, we reported our effort to conduct a structure-based optimization of NSD2-PWWP1 ligands, leading to a selective NSD2-PWWP1 inhibitor 38 with significantly improved potency. Compound 38 can clearly bind to NSD2-PWWP1 and further affect the expression of genes regulated by NSD2. We believe that the discovery and related biological evaluation will contribute to the further research of the specific regulation mode of NSD2 by PWWP1 recognition, and furthermore, our discovery also provided a certain material basis for the functional study of NSD2.