The ubiquitin–proteasome system (UPS) represents one of the most promising therapeutic targets in oncology to emerge in recent years. Here we used a combination of cytotoxic and image-based screening assays to identify a novel UPS inhibitor, designated HRF-3. HRF-3 evokes a gene expression profile similar to that of other characterized UPS inhibitors, suggesting a common mechanism of action. Consistent with UPS inhibition, HRF-3 induced strong accumulation of polyubiquitinated proteins in cells. Surprisingly, HRF-3 induced only weak accumulation of two proteasome targeted reporter proteins, UbG76V-YFP and ZsGreen-ODC. Consistent with this observation, HRF-3 did not inhibit proteasome proteolytic activity in an in vitro assay. Similar to a number of other UPS inhibitors, HRF-3 increased the expression of the redox-inducible protein Hmox-1. In distinction to the 20S inhibitor bortezomib, but similarly to two different p97/VCP inhibitors, HRF-3 did not elicit strong induction of the chaperone Hsp70B0. Finally, we show that HRF-3 is cytotoxic to a variety of cancer cell lines and ex vivo patient tumour cells, with the strongest activity observed in cells of leukemic/myeloma origin. Taken together our data show that HRF-3 induces polyubiquitin accumulation in the absence of efficient proteasomal blocking, and suggest that induction of oxidative stress is a common denominator of UPS inhibitors.