L-threonine is an important amino acid that can be added in food, medicine, or feed. Here, the influence of glyoxylate shunt on an l-threonine producing strain Escherichia coli TWF001 has been studied. The gene iclR was deleted, and the native promoter of the aceBA operon was replaced by the trc promoter in the chromosome of TWF001, the resulting strainTWF004 could produce 0.39 g l-threonine from1 g glucose after 36-h flask cultivation. Further replacing the native promoter of aspC by the trc promoter in the chromosome of TWF004 resulted in the strain TWF006. TWF006 could produce 0.42 g l-threonine from 1 g glucose after 36-h flask cultivation. Three key genes in the biosynthetic pathway of l-threonine, thrA (*) (a mutated thrA), thrB, and thrC were overexpressed in TWF006, resulting the strain TWF006/pFW01-thrA (*) BC. TWF006/pFW01-thrA (*) BC could produce 0.49 g l-threonine from 1 g glucose after 36-h flask cultivation. Next, the genes asd, rhtA, rhtC, or thrE were inserted into the plasmid TWF006/pFW01-thrA (*) BC, and TWF006 was transformed with these plasmids, resulting the strains TWF006/pFW01-thrA (*) BC-asd, TWF006/pFW01-thrA (*) BC-rhtA, TWF006/pFW01-thrA (*) BC-rhtC, and TWF006/pFW01-thrA (*) BC-thrE, respectively. These four strains could produce more l-threonine than the control strain, and the highest yield was produced by TWF006/pFW01-thrA (*) BC-asd; after 36-h flask cultivation, TWF006/pFW01-thrA (*) BC-asd could produce 15.85 g/l l-threonine, i.e., 0.53 g l-threonine per 1 g glucose, which is a 70% increase relative to the control strain TWF001. The results suggested that the combined engineering of glyoxylate shunt and l-threonine biosynthesis pathway could significantly increase the l-threonine production in E. coli.