Effective inhibition of the proteasome by high doses of proteasome inhibitors induces apoptotic cell death. In contrast, partial proteasome inhibition by low inhibitor doses mediates a protective cellular stress response. The early targets and mediators of these dose-dependent effects of proteasome inhibitors are unknown. Primary human umbilical cord vein endothelial cells were treated with low and high doses of the proteasome inhibitor MG132 for 2 h. In a combined 2-DE and MS approach, we identified more than 20 new targets of proteasome inhibition. These proteins are involved in cell cycle regulation, signaling, cytoskeletal rearrangement, and cellular stress response. Accompanying Affymetrix analysis revealed that these proteins are not regulated on the transcriptional level but are mainly stabilized by proteasome inhibition. The proteasome-dependent accumulation of the anti-oxidative sensor proteins DJ-1, peroxiredoxin-1 and -6 was accompanied by dose-dependent induction of oxidative stress after 2 h of proteasome inhibition and contributed to the differential transcriptional stress response to low- and high-dose proteasome inhibition: Whereas low-dose proteasome inhibition induces a transcriptional profile reminiscent of a physiological stress response that preconditions and protects endothelial cells from oxidative stress, high inhibitor doses induce massive transcriptional dysregulation and pronounced oxidative stress triggering apoptosis.