Protein ubiquitination via the covalent attachment of ubiquitin (Ub) plays an important role in the regulation of the stability, function or localization of multiple proteins in eukaryotic cells. Comprehensive investigation of the proteomics related to ubiquitination will gain the insight into the Ub-mediated regulatory mechanism. In the present study, the combination of polyUb affinity purification, SDS-PAGE separation, and liquid chromatography-tandem mass spectrometry analysis (GeLC-MS/MS) was employed to analyze the Ub-related proteins in human MDA-MB-231 breast carcinoma epithelial cells after treatment with the proteasome inhibitor MG132. A total of 260 non-redundant Ub-related proteins were identified from the cells. These proteins were shown to be involved in a host of critical cellular functions and processes, including transcription, translation, Ub-proteasome pathway, cell cycle, heat shock response, transport, etc. The interaction network analysis by STRING indicated that the identified Ub-related proteins formed eleven clusters, the three most highly ranked network clusters were mainly involved in protein translation, RNA transcription and processing, and Ub-proteasome pathway, suggesting that there were obvious ubiquitination-mediated alternations in gene expression of human MDA-MB-231 cells. The proteomic profiling and their interaction network analysis in this study would help to our systematic understanding of the Ub-related cellular protein functions and the related biological processes in human disease tissue cells.