Mono-, di- and triacetin are three glycerol esters which are usually synthesized via costly and environmentally unfriendly chemical synthesis methods. Here, Escherichia coli is metabolically engineered for the production of mono-, di- and triacetin using glycerol as a substrate. First, a novel biosynthetic route of mono- and diacetin is established by overexpression of a native enzyme, maltose O-acetyltransferase (MAA). Next, the biosynthetic pathway is extended to produce a mixture of mono-, di- and triacetin by overexpression of chloramphenicol-O-acetyltransferase (CAT). By successive strategies, including heterologous gene expression, metabolic engineering, and culture optimization, a recombinant E. coli is enabled to produce more than 27 g L⁻¹ of a mixture of mono-, di- and triacetin in shake flask cultures, which is a >650-fold increase over the initial production of 0.04 g L⁻¹. In vitro studies confirm the acetylation of glycerol to mono- and diacetin by MAA, and the additional acetylation to triacetin by CAT. When crude glycerol is used as a substrate, the engineered strain produced a total of 25.9 g L⁻¹ of the acetin mixture, about the same as that achieved from pure glycerol. To our knowledge, this is the first successful report of microbial production of the artificial chemicals, acetins.