The stress response gene IEX-1 (immediate early gene-X-1) is involved in the regulation of cell growth and cellular viability. To some extent, these effects include an interference with the proteasomal turnover of certain regulatory proteins. Here, we show that IEX-1 directly attenuates the activity and formation of the 26 S proteasome in HEK-293 cells (human embryonic kidney cells). We further demonstrate that IEX-1 reduces the overall expression levels of certain protein components of the 19 S proteasomal subunit such as S5a/Rpn10 and S1/Rpn2, whereas the expression of other proteasomal proteins was less or not affected. In contrast with direct apoptotic stimuli, such as the anti-cancer drug etoposide, leading to caspase-dependent degradation of S1 and S5a, the effect of IEX-1 is independent of proteolytic cleavage of these proteins. Furthermore, the decreasing effect of IEX-1 on S5a and S1 expression is still seen in the presence of cycloheximide, but not in the presence of actinomycin D, and quantitative real-time PCR revealed lower mRNA levels of S5a and S1 in IEX-1-overexpressing cells, suggesting an interference of IEX-1 with the gene transcription of S5a and S1. Additionally, luciferase assays confirmed an interference of IEX-1 with the activity of the S5a promoter. These findings indicate a role of IEX-1 in the maintenance and assembly of the 26 S proteasome, obviously involving an altered gene expression of certain proteasomal proteins. Thereby, IEX-1 may essentially modulate signalling pathways related to 26 S proteasome activity and involved in cellular growth control and apoptosis.