Abstract: OBJECTIVE To design and synthesize a series of non-covalent peptidomimetic proteasome inhibitors, and to evaluate their enzymatic activities. METHODS A series of non-covalent short peptidyl proteasome inhibitors were designed and synthesized with rational drug design strategies such as bioisostere and amino acid replacement based on the binding modes of the non-covalent proteasome inhibitors with the proteasome. 2-Chloride benzyl amine was selected as the carboxyl terminal group, and a six-membered ring was introduced into the peptide skeleton to increase the compounds' stability. All the target compounds were tested for their enzymatic inhibitory activities in vitro. RESULTS Totally 8 novel di-and tripeptidyl analogues were synthesized, which were confirmed by ¹H-NMR and ESI-MS. These target compounds exhibited moderate proteasome inhibitory activities. CONCLUSION The proteasome inhibitory activities of the target compounds are influenced by the length of the peptides and various amino terminal substituents. All the 8 compounds display different degree of proteasome inhibitory activities in vitro. In a word, this study develops series of proteasome inhibitors with new structure type, which will be helpful for the further development of this series of compounds.