[Background] Escherichia coli BL21(DE3) is a commonly used host for genetic engineering. It synthesizes 5-aminolevulinic (ALA) through C5 pathway. ALA is an important precursor, while its secretion on the heme synthesis is unclear. [Objective] To elucidate the role of RhtA in the export of ALA in the heme synthesis pathway. [Methods] rhtA was knocked out by Red homologous recombination, and plasmid pEA was constructed to overexpress hemA, a key enzyme gene in the heme biosynthesis pathway. The contents of heme and its precursors as well as the transcription levels of 10 key genes in the heme synthesis pathway were analyzed. [Results] The deletion of rhtA had no significant effect on cell growth. Compared with parental strain BL21(DE3), the extracellular content of ALA decreased by 23% while the content of heme increased by 12% in the knockout strain BL21(DE3)ΔrhtA. The contents of uroporphyrin III (UIII), coproporphyrin III (CIII) and protoporphyrin IX (PPIX) increased by 25%, 15% and 18%, respectively in BL21(DE3)ΔrhtA. Compared with the strain BL21(DE3)/pEA in which hemA was overexpressed, the content of extracellular ALA reduced by 16% and the content of heme increased by 24% in the strain BL21(DE3)ΔrhtA/pEA. And the contents of UIII and CIII increased by 55% and 64%, respectively. The content of PPIX increased significantly, about 4.7 times in BL21(DE3)ΔrhtA/pEA compared with that in BL21(DE3)/pEA. The results of real-time quantitative PCR showed that after the deletion of rhtA, the transcription level of hemC was down-regulated, while those of the other 9 genes were up-regulated to various degrees. [Conclusion] The rhtA knockout reduces the export of ALA and leads to an increase in intracellular heme production.