Proteasome inhibition represents a promising strategy of cancer pharmacotherapy, but resistant tumor cells often emerge. Here we show that the microRNA-101 (miR-101) targets the proteasome maturation protein POMP, leading to impaired proteasome assembly and activity, and resulting in accumulation of p53 and cyclin-dependent kinase inhibitors, cell cycle arrest, and apoptosis. miR-101-resistant POMP restores proper turnover of proteasome substrates and re-enables tumor cell growth. In ERα-positive breast cancers, miR-101 and POMP levels are inversely correlated, and high miR-101 expression or low POMP expression associates with prolonged survival. Mechanistically, miR-101 expression or POMP knockdown attenuated estrogen-driven transcription. Finally, suppressing POMP is sufficient to overcome tumor cell resistance to the proteasome inhibitor bortezomib. Taken together, proteasome activity can not only be manipulated through drugs, but is also subject to endogenous regulation through miR-101, which targets proteasome biogenesis to control overall protein turnover and tumor cell proliferation.