Protein degradation by the ubiquitin‐proteasome pathway plays an important role in a variety of fundamental cellular processes, including cell cycle regulation, transcription, antigen processing and muscle remodelling. Research into disorders associated with the ubiquitin‐proteasome system has been mainly in the field of neurodegenerative diseases. It is however becoming increasingly apparent that defects in the system are responsible for a number of non‐neurological pathologies. Based on initial observations made as part of a proteomic analysis of an animal model of dilated cardiomyopathy (DCM) which indicated increased activity of the ubiquitin‐proteasome system, we sought to determine whether this system was perturbed in hearts of human DCM patients. We studied explanted hearts from 12 DCM, 9 ischaemic (IHD) and 12 unused donor hearts. Protein expression was examined using two‐dimensional polyacrylamide gel electrophoresis, Western blotting and immunohistochemistry. Expression of mRNA was examined using real‐time quantitative polymerase chain reaction. Ubiquitinated proteins were affinity purified using a ubiquitin‐binding column and identified using peptide mass fingerprinting. All DCM hearts showed significantly higher expression of certain key enzymes of the ubiquitin‐proteasome pathway. mRNA expression of ubiquitin carboxyl‐terminal hydrolase (UCH) was significantly higher (5.4‐fold) in DCM hearts than in control hearts. Myocytes in sections from DCM hearts stained positively for UCH, whereas control hearts were negative. Overall protein ubiquitination was increased two‐fold in DCM relative to IHD hearts and five‐fold relative to donor hearts. The ubiquitination of a number of distinct proteins was greatly enhanced in DCM hearts as revealed by anti‐ubiquitin Western blots. A number of these proteins were identified using affinity purification and matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry.