Substitution of protium (H) for deuterium (D) strongly affects biological systems. Whereas higher eukaryotes such as plants and mammals hardly survive a deuterium content of >30%, many microorganisms can grow on fully deuterated media, albeit at reduced rates. Very little is known about how the H/D replacement influences life at the systems level. Here, we used MS-based analysis to follow the adaptation of a large part of the Escherichia coli proteome from growth on a protonated full medium, over a protonated minimal medium, to a completely deuterated minimal medium. We could quantify >1800 proteins under all conditions, several 100 of which exhibited strong regulation during both adaptation processes. The adaptation to minimal medium strongly up-regulated amino acid synthesis and sugar metabolism and down-regulated translational proteins on average by 9%, concomitant with a reduction in growth rate from 1.8 to 0.67 h-1. In contrast, deuteration caused a very wide proteomic response over many cell functional categories, together with an additional down-regulation of the translational proteins by 5%. The latter coincided with a further reduction in growth rate to 0.37 h-1, revealing a clear linear correlation between growth rate and abundance of translational proteins. No significant morphological effects are observed under light and electron microscopies. Across all protein categories, about 80% of the proteins up-regulated under deuteration are enzymes with hydrogen transfer functions. Thus, the H/D kinetic isotope effect appears as the major limiting factor of cellular functions under deuteration.