The productivity of Escherichia coli as a producer of recombinant proteins is affected by its metabolic properties, especially by acetate production. Two commercially used E. coli strains, BL21 (λDE3) and JM109, differ significantly in their acetate production during batch fermentation at high initial glucose concentrations. E. coli BL21 grows to an optical density (OD, 600 nm) of 100 and produces no more than 2 g/L acetate, while E. coli JM109 grows to an OD (600 nm) of 80 and produces up to 14 g/L acetate. Even in fed-batch fermentation, when glucose concentration is maintained between 0.5 and 1.0 g/L, JM109 accumulates 4 times more acetate than BL21. To investigate the difference between the two strains, metabolites and enzymes involved in carbon utilization and acetate production were analyzed (isocitrate, ATP, phosphoenolpyruvate, pyruvate, isocitrate lyase, and isocitrate dehydrogenase). The results showed that during batch fermentation isocitrate lyase activity and isocitrate concentration were higher in BL21 than in JM109, while pyruvate concentration was higher in JM109. The activation of the glyoxylate shunt pathway at high glucose concentrations is suggested as a possible explanation for the lower acetate accumulation in E. coli BL21. Metabolic flux analysis of the batch cultures supports the activity of the glyoxylate shunt in E. coli BL21.