Reversible lysine acetylation (RLA) of translation machinery components, such as ribosomal proteins (RPs) and translation factors (TFs), was identified in many microorganisms, while knowledge of its function and effect on translation remains limited. Herein, we show that translation machinery is regulated by acetylation. Using the cell-free translation system of E. coli, we found that AcP-driven acetylation significantly reduced the relative translation rate, and deacetylation partially restored the translation activity. Hyperacetylation caused by intracellular AcP accumulation or carbon/nitrogen fluctuation (carbon overflow or nitrogen limitation) modulated protein translation in vivo. These results uncovered a critical role of acetylation in translation regulation and indicated that carbon/nitrogen imbalance induced acetylation of ribosome in E. coli and dynamically affected translation rate via a global, uniform manner.