A series of inhibitors of 20S proteasome based on terminal functionalized dipeptide derivatives containing the thiourea moiety were synthesized and evaluated for inhibition of 20S proteasome and the effects of multidrug-resistance reversers. These compounds exhibited significant selectivity to the β5-subunit of the human 20S proteasome with IC50 values at submicromolar concentrations. A docking study of the most active compound 6i revealed key interactions between 6i and the active site of the 20S proteasome in which the thiourea moiety and a nitro group were important for improving activity. In particular, compound 6i appeared to be the most potent compound against the NCI-H460 cell line, and displayed similar efficiency in drug-sensitive versus drug-resistant cancer cell lines, at least partly, by inhibition of the activity of 20S proteasome and induce apoptosis. In addition, 6i-induced apoptosis was significantly facilitated in NCI-H460/DOX cells that had been pretreated with inhibitors of P-gp. Mechanistically, compound 6i might trigger apoptotic signalling pathway. Thus, we conclude that dipeptide derivatives containing the thiourea moiety may be the potential inhibitors of proteasome with the ability to reverse multidrug resistance.