Neddylation of the Cullin-RING E3 ligases (CRLs) regulates the homeostasis of approximately 20% of cellular proteins. Defective in cullin neddylation 1 (DCN1), as a co-E3 ligase, interacts with UBE2M to enhance the activation of CRLs, and this interaction is emerging as a therapeutic target for human diseases. Here, we present a series of pyrimidin-based small molecular inhibitors targeting DCN1-UBE2M interaction. After finding a novel inhibitor DC-1 with IC50 = 1.2 μM, we performed a series of chemical optimizations, which finally led to the discovery of a potent thiazole containing 5-cyano-6-phenylpyrimidin-based inhibitor DC-2 (IC50 = 15 nM). Next, using protein and cellular thermal shift assays, coimmunoprecipitation, molecular docking, and site-specific mutation experiments, we further proved that DC-2 specifically inhibited the interaction of UBE2M and DCN1 at molecule and cellular levels, resulting in the decrease of cullin3 neddylation and accumulation of its substrate, NRF2. Our findings indicate that DC-2 may serve as a novel lead compound for specific derivatives targeting DCN1-UBE2M interaction.