ABSTRACT 2′‐Fucosyllactose (2‐FL) is one of the key oligosaccharides in human milk. In the present study, the salvage guanosine 5′‐diphosphate (GDP)‐l‐fucose biosynthetic pathway from fucose was employed in engineered Escherichia coli BL21star(DE3) for efficient production of 2‐FL. Introduction of the fkp gene coding for fucokinase/GDP‐l‐fucose pyrophosphorylase (Fkp) from Bacteroides fragilis and the fucT2 gene encoding α‐1,2‐fucosyltransferase from Helicobacter pylori allows the engineered E. coli to produce 2‐FL from fucose, lactose and glycerol. To enhance the lactose flux to 2‐FL production, the attenuated, and deleted mutants of β‐galactosidase were employed. Moreover, the 2‐FL yield and productivity were further improved by deletion of the fucI‐fucK gene cluster coding for fucose isomerase (FucI) and fuculose kinase (FucK). Finally, fed‐batch fermentation of engineered E. coli BL21star(DE3) deleting lacZ and fucI‐fucK, and expressing fkp and fucT2 resulted in 23.1 g/L of extracellular concentration of 2‐FL and 0.39 g/L/h productivity. Biotechnol. Bioeng. 2016;113: 2443–2452. © 2016 Wiley Periodicals, Inc. 2’‐Fucosyllactose (2‐FL) is one of the main oligosaccharides in human milk. The authors engineered Escherichia coli to produce 2‐FL from fucose and lactose. The key strategies include introduction of the fucokinase/GDP‐l‐fucose pyrophosphorylase (Fkp) from Bacteroides fragilisacteroides fragilisandtheα‐1,2‐fucosyltransferase (FucT2) from Helicobacter pylorielicobacter pylori, anddeletion of thegenes involved in lactose and fucose metabolisms. The fed‐batch fermentation of the final strain resulted in 23.1 g/L of 2‐FL.