[Background] Escherichia coli uses glutamic acid as a precursor to synthesize limited heme through the C5 pathway. [Objective] The purpose of this study was to investigate the effects of the regulation of intracellular glutamic acid metabolism and over-expression of glutamyl-tRNA reductase gene (hemA) on 5-aminolevulinic acid (ALA) and heme synthesis. [Methods] Through Red homologous recombination, mscS and aroG which are related to glutamate metabolism were knocked out, and the hemA expression vector was constructed and introduced into the gene-knockout strains. [Results] The single and double gene-knockout had no significant effect on cell growth, but the content of glutamic acid increased, whereas that of ALA decreased slightly. The heme content in single and double gene-knockout strains increased by 11.6% and 35.7% compared with that in the parental strain respectively. [Conclusion] The over-expression of hemA in the double gene-knockout strain caused the increase of heme to 47.603 μmol/L. Heme synthesis can be promoted by regulation of intracellular glutamate metabolic flow and over-expression of hemA which provides a new approach for heme enhancement through C5 pathway.