Substitution of hydrogen for deuterium strongly affects biological systems. While higher eukaryotes, such as plants and mammals, hardly survive >30% deuterium content of water and nutrients, many microorganisms can grow on fully deuterated media albeit at reduced rates. Due to the large relative mass change, H/D replacement leads to pronounced changes in chemical reaction rates and equilibria. Very little is known how these physico-chemical effects influence cellular life at the systems level. Here we have followed the adaptation of a large part of the E. coli proteome from growth on a protonated full medium, over a protonated minimal to a completely deuterated minimal medium. More than 1800 proteins could be quantified under all conditions and several hundreds show strong regulation in both adaptation processes. As expected, the adaption to minimal medium mostly upregulates amino acid synthesis and sugar metabolism. In contrast, deuteration causes a very wide response over many cell function categories. No morphological effects are apparent under light and electron microscopy. Most of the regulated proteins have enzymatic functions involving hydrogen transfer reactions. This indicates that kinetic isotope effects and not changes in biomolecular stability are the dominant mechanisms that affect cellular function under deuteration.