1,2,4-Oxadiazole derivatives, a class of Nrf2-ARE activators, exert an extensive therapeutic effect on inflammation, cancer, neurodegeneration, and microbial infection. Among these analogues, <b>DDO-7263</b> is the most potent Nrf2 activator and used as the core structure for bioactive probes to explore the precise mechanism. In this work, we obtained compound <b>7</b>, a mimic of <b>DDO-7263</b>, and biotin-labeled and fluorescein-based probes, which exhibited homologous biological activities to <b>DDO-7263</b>, including activating Nrf2 and its downstream target genes, anti-oxidative stress, and anti-inflammatory effects. Affinity chromatography and mass analysis techniques revealed Rpn6 as the potential target protein regulating the Nrf2 signaling pathway. In vitro affinity experiments further confirmed that <b>DDO-7263</b> upregulated Nrf2 through binding to Rpn6 to block the assembly of 26S proteasome and the subsequent degradation of ubiquitinated Nrf2. These results indicated that Rpn6 is a promising candidate target to activate the Nrf2 pathway for protecting cells and tissues from oxidative, electrophilic, and exogenous microbial stimulation.