AbstractStudy of the complexome – all the protein complexes of the cell – is essential for a better understanding and more global vision of cell function. Using two‐dimensional blue native/SDS‐PAGE (2‐D BN/SDS‐PAGE) technology, the cytosolic and membrane protein complexes of Escherichia coli were separated. Then, the different partners of each protein complex were identified by LC‐MS/MS. In this report, 306 protein complexes were separated and identified. Among these protein complexes, 50 heteromultimeric and 256 homomultimeric protein complexes were found. Among the 50 heteromultimeric protein complexes, 18 previously described protein complexes validate the technology. In this study, 109 new protein complexes were found, providing insight into the function of previously uncharacterized bacterial proteins.