Microbial production of 5-aminolevulinic acid (ALA) has received much attention because of its potential in clinical applications. Overexpression of related enzymes and an transporter yielded remarkable achievements in ALA production. Nonetheless, there is significant room for carbon flux optimization to enhance ALA production. The aim of this study was precise carbon flux optimization for high ALA production in Escherichia coli. Initially, genes hemA and hemL were overexpressed with strong promoters and synthetic 5'-untranslated regions (5'-UTRs). Then, TCA cycle was blocked to force carbon flux toward the ALA production pathway by deletion of sucA. In spite of metabolic imbalance in the strain, further precise tuning of carbon flux to the glyoxylate cycle by varying the transcriptional strength of aceA led to substantially improved cell growth and ALA production.