Inter-species interactions shape the function and dynamics of microbial communities. Yet, the understanding of how these interactions influence the molecular physiology of interacting species remains surprisingly limited. Here, we assembled 104 synthetic pairwise co-cultures of 15 diverse gut bacteria in vitro and examined responses using meta-proteomics and metabolomics. We detect a diversity of ecological interactions with an overall dominance of competition, amensalism and exploitation, yet with a circa 18% overall prevalence of positive interactions. The growth changes were accompanied by strong molecular changes, with typically more than 60% of the proteome of a species changing in response to at least one partner. We find that the magnitude of the proteome responses is influenced by proteome size, species abundance, and pH of the co-culture. Small-molecule transport and carbon metabolism are among the most responsive processes indicating pervasive metabolic interactions. Using metabolomics we identified likely cross-fed metabolites, emergent polyamine metabolism, and niche partitioning in amino acid utilization in co-cultures. Notably, many yet uncharacterized proteins were differentially abundant in co-cultures, suggesting a role of the ‘dark proteome’ in inter-species interactions. Clustering of these proteins with those with known biological function indicates an approach to leverage species interactions for functional genomics. Overall, our systematic approach demonstrates how bacterial interactions profoundly affect the physiology of participating cells, advancing our molecular understanding of ecological interactions.