Acetate is ubiquitously found in natural environments. Its availability in the gut is high as a result of the fermentation of nutrients, and although it is rapidly absorbed by intestinal mucosa, it can also be used as carbon source by some members of gut microbiota. The metabolism of acetate in Escherichia coli has attracted the attention of the scientific community due to its role in central metabolism and its link to multiple physiological features. In this microorganism, acetate is involved directly or indirectly on the regulation of functional processes, such as motility, formation of biofilms, and responses to stress. Furthermore, it is a relevant nutrient in gut, where it serves additional roles, which regulate or, at least, modulate pathophysiological responses of E. coli and other bacteria. Acetate is one of the major by-products of anaerobic (fermenting) metabolism, and it is also produced under fully aerobic conditions. This acetate overflow is recognized as one of the major drawbacks limiting E. coli's productivity in biotechnological processes. This review sums up current knowledge on acetate metabolism in E. coli, explaining the major milestones that have led to deciphering its complex regulation in the K-12 strain. Major differences in the metabolism of acetate in other strains will be underlined, with a focus on strains of biotechnological and biomedical interest.