The first-generation enhancer of zeste homologue 2 (EZH2) inhibitors suffer from several limitations, such as high dosage, cofactor S-adenosylmethionine (SAM) competition, and acquired drug resistance. Development of covalent EZH2 inhibitors that are noncompetitive with cofactor SAM offers an opportunity to overcome these disadvantages. The structure-based design of compound 16 (BBDDL2059) as a highly potent and selective covalent inhibitor of EZH2 is presented in this context. 16 inhibits EZH2 enzymatic activity at sub-nanomolar concentrations and achieves low nanomolar potencies in cell growth inhibition. The kinetic assay revealed that 16 is noncompetitive with the cofactor SAM, providing the basis for its superior activity over noncovalent and positive controls by reducing competition with cofactor SAM and offering a preliminary proof for its covalent inhibition nature. Mass spectrometric analysis and washout experiments firmly establish its covalent inhibition mechanism. This study demonstrates that covalent inhibition of EZH2 can offer a new opportunity for the development of promising new-generation drug candidates.