The protein–protein interaction between menin and mixed-lineage leukemia 1 (MLL1) plays an important role in development of acute leukemia with translocations of the MLL1 gene and in solid tumors. Here, we report the development of a new generation of menin–MLL1 inhibitors identified by structure-based optimization of the thienopyrimidine class of compounds. This work resulted in compound 28 (MI-1481), which showed very potent inhibition of the menin–MLL1 interaction (IC₅₀ = 3.6 nM), representing the most potent reversible menin–MLL1 inhibitor reported to date. The crystal structure of the menin-28 complex revealed a hydrogen bond with Glu366 and hydrophobic interactions, which contributed to strong inhibitory activity of 28. Compound 28 also demonstrates pronounced activity in MLL leukemia cells and in vivo in MLL leukemia models. Thus, 28 is a valuable menin–MLL1 inhibitor that can be used for potential therapeutic applications and in further studies regarding the role of menin in cancer.