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L‐Threonine, a kind of essential amino acid, has numerous applications in food, pharmaceutical, and aquaculture industries. Fermentative l‐threonine production from glucose has been achieved in Escherichia coli. However, there are still several limiting factors hindering further improvement of l‐threonine productivity, such as the conflict between cell growth and production, byproduct accumulation, and insufficient availability of cofactors (adenosine triphosphate, NADH, and NADPH). Here, a metabolic modification strategy of two‐stage carbon distribution and cofactor generation was proposed to address the above challenges in E. coli THRD, an l‐threonine producing strain. The glycolytic fluxes towards tricarboxylic acid cycle were increased in growth stage through heterologous expression of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and citrate synthase, leading to improved glucose utilization and growth performance. In the production stage, the carbon flux was redirected into l‐threonine synthetic pathway via a synthetic genetic circuit. Meanwhile, to sustain the transaminase reaction for l‐threonine production, we developed an l‐glutamate and NADPH generation system through overexpression of glutamate dehydrogenase, formate dehydrogenase, and pyridine nucleotide transhydrogenase. This strategy not only exhibited 2.02‐ and 1.21‐fold increase in l‐threonine production in shake flask and bioreactor fermentation, respectively, but had potential to be applied in the production of many other desired oxaloacetate derivatives, especially those involving cofactor reactions. L‐Threonine, a kind of essential amino acid, has numerous applications in food, pharmaceutical. and aquaculture industries. Fermentative l‐threonine production from glucose has been achieved in Escherichia coli. However, there are still several limiting factors hindering further improvement of l‐threonine productivity, such as the conflict between cell growth and production, byproduct accumulation, and insufficient availability of cofactors (adenosine triphosphate, NADH, and NADPH). [ABSTRACT FROM AUTHOR]