The ubiquitin proteasome system (UPS) degrades abnormal proteins and most unneeded normal proteins, thereby playing a critical role in protein homeostasis in the cell. Proteasome inhibition is effective in treating certain forms of cancer while UPS dysfunction is increasingly implicated in the pathogenesis of many severe and yet common diseases. It was previously shown that doxorubicin (Dox) enhances the degradation of a UPS surrogate substrate in mouse hearts. To address the underlying mechanism, here we report that: (1) Dox not only enhances the degradation of an exogenous UPS reporter (GFPu) but also antagonizes proteasome inhibitor induced accumulation of endogenous substrates (e.g., -catenin, c-Jun) of the UPS in cultured NIH 3T3 cells and cardiomyocytes; (2) Dox facilitates in vitro degradation of GFPu and c-Jun by the reconstituted UPS via enhancing proteasomal function; (3) Dox at a therapeutically relevant dose directly stimulates the peptidase activities of purified 20S proteasomes; and (4) Dox increases, while proteasome inhibition decreases, E3 ligase CHIP in 3T3 cells via a post-transcriptional mechanism. These new findings suggest that Dox activates the UPS by acting directly on both the ubiquitination apparatus and the proteasome.