Abstract Protein interactions are essential elements for the biological machineries underlying biochemical and physiological mechanisms indispensable for microorganism life. By using mono-dimensional blue native polyacrylamide gel electrophoresis (1D-BN-PAGE), two-dimensional blue native/urea-PAGE (2D-BN/urea-PAGE) and two-dimensional blue native/SDS-PAGE (2D-BN/SDS-PAGE), membrane protein complexes of Streptococcus thermophilus were resolved and visualized. Protein complex and oligomer constituents were then identified by nLC–ESI-LIT-MS/MS. In total, 65 heteromeric and 30 homomeric complexes were observed, which were then associated with 110 non-redundant bacterial proteins. Protein machineries involved in polysaccharide biosynthesis, molecular uptake, energy metabolism, cell division, protein secretion, folding and chaperone activities were highly represented in electrophoretic profiles; a number of homomeric moonlighting proteins were also identified. Information on hypothetical proteins was also derived. Parallel genome sequencing unveiled that the genes coding for the enzymes involved in exopolysaccharide biosynthesis derive from two separate clusters, generally showing high variability between bacterial strains, which contribute to a unique, synchronized and active synthetic module. The approach reported here paves the way for a further functional characterization of these protein complexes and will facilitate future studies on their assembly and composition during various growth conditions and in different mutant backgrounds, with important consequences for biotechnological applications of this bacterium in dairy productions. Biological significance Combined proteomic procedures have been applied to the characterization of heteromultimeric and homomeric protein complexes from the membrane fraction of S. thermophilus. Protein machineries involved in polysaccharide biosynthesis, molecular uptake, energy metabolism, cell division, protein secretion, folding and chaperone activities were identified; information on hypothetical and moonlighting proteins were also derived. This study is original in the lactic bacteria context and may be considered as preliminary to a deeper functional characterization of the corresponding protein complexes. Due to the large use of S. thermophilus as a starter for dairy productions, the data reported here may facilitate future investigations on protein complex assembly and composition under different experimental conditions or for bacterial strains having specific biotechnological applications.