We demonstrate that rebalancing of metabolic fluxes at acetyl-CoA can substantially improve the titer and productivity of hexanoic acid. First, a hexanoic acid-producing E. coli strain was constructed by expressing the genes coding β-ketothiolase (BktB) from Cupriavidus necator and acetyl-CoA transferase (ACT) from Megasphaera sp. MH in a butyric acid producer strain. Expression of mhact coding ACT increased hexanoic acid production by 47% compared to that with an E. coli thioesterase, TesB. Next, metabolic flux was optimized at the acetyl- CoA branch point by fine-tuning the expression level of the gene for acetyl-CoA acetyltransferase (AtoB). Five different synthetic 5′ -untranslated regions (5′-UTRs) were designed for atoB using UTR Designer to modulate the expression level of the gene. The fluxoptimized variant produced 528 mg/L hexanoic acid in 36 h, and this titer represents an 8.7-fold increase compared to that of the nonoptimized parental strain.