Palbociclib is a CDK4/6 inhibitor approved for metastatic estrogen receptor‐positive breast cancer. In addition to G1 cell cycle arrest, palbociclib treatment results in cell senescence, a phenotype that is not readily explained by CDK4/6 inhibition. In order to identify a molecular mechanism responsible for palbociclib‐induced senescence, we performed thermal proteome profiling of MCF7 breast cancer cells. In addition to affecting known CDK4/6 targets, palbociclib induces a thermal stabilization of the 20S proteasome, despite not directly binding to it. We further show that palbociclib treatment increases proteasome activity independently of the ubiquitin pathway. This leads to cellular senescence, which can be counteracted by proteasome inhibitors. Palbociclib‐induced proteasome activation and senescence is mediated by reduced proteasomal association of ECM29. Loss of ECM29 activates the proteasome, blocks cell proliferation, and induces a senescence‐like phenotype. Finally, we find that ECM29 mRNA levels are predictive of relapse‐free survival in breast cancer patients treated with endocrine therapy. In conclusion, thermal proteome profiling identifies the proteasome and ECM29 protein as mediators of palbociclib activity in breast cancer cells. Synopsis The breast cancer drug palbociclib arrests cells in G1 phase by CDK4/6 inhibition, but also causes cellular senescence. Thermal proteome profiling shows that the latter is mediated by increased proteasome activation through reduced ECM29 binding. Mass spectrometry‐based cellular thermal shift assay (MS‐CeTSA) analysis of CDK4/6 inhibitor palbociclib targets in MCF7 human breast cancer cells identifies protein complexes including the 20S proteasome. Palbociclib treatment increases proteasome activity independently of the ubiquitin pathway. Palbociclib activates the proteasome by disassociating the proteolysis‐inhibiting scaffold protein ECM29. Proteasome activation is required for palbociclib‐induced cellular senescence, which is sensitive to the proteasome inhibitor bortezomib. The proteasome is a novel downstream target for the anti‐cancer drug palbociclib inhibiting proliferation by reduced proteasomal association with its inhibitor EMC29.