AbstractProteins play an essential role in the vital biological processes governing cellular functions. Most proteins function as members of macromolecular machines, with the network of interacting proteins revealing the molecular mechanisms driving the formation of these complexes. Profiling the physiology‐driven remodeling of these interactions within different contexts constitutes a crucial component to achieving a comprehensive systems‐level understanding of interactome dynamics. Here, we apply co‐fractionation mass spectrometry and computational modeling to quantify and profile the interactions of ∼2000 proteins in the bacterium Escherichia coli cultured under 10 distinct culture conditions. The resulting quantitative co‐elution patterns revealed large‐scale condition‐dependent interaction remodeling among protein complexes involved in diverse biochemical pathways in response to the unique environmental challenges. The network‐level analysis highlighted interactome‐wide biophysical properties and structural patterns governing interaction remodeling. Our results provide evidence of the local and global plasticity of the E. coli interactome along with a rigorous generalizable framework to define protein interaction specificity. We provide an accompanying interactive web application to facilitate the exploration of these rewired networks.