There is an urgent need for new tools that enable better understanding of the structure, recognition, metabolism, and biosynthesis of glycans as well as the production of biologically important glycans and glycoconjugates. With the discovery of glycoprotein synthesis in bacteria and functional transfer of glycosylation pathways between species, Escherichia coli cells have become a tractable host for both understanding glycosylation and the underlying glycan code of living cells as well as for expressing glycoprotein therapeutics and vaccines. Here, we review recent efforts to harness natural biological pathways and engineer synthetic designer pathways in bacteria for making complex glycans and conjugating these to lipids and proteins. The result of these efforts has been a veritable transformation of bacteria into living factories for scalable, bottom-up production of complex glycoconjugates by design. Biotechnol. Bioeng. 2013; 110: 1550–1564. © 2013 Wiley Periodicals, Inc.