Shikimic acid is one of the most important chiral compounds synthesized in the aromatic amino acid pathway of plants and microorganisms. It is mostly used to produce Tamiflu to treat avian flu infections. In E. coli, shiA gene is responsible for shikimic acid uptake from the culture media. In this work, shA gene has been knocked out in E. coli JM83 and JDL02 to develop new strains of E. coli JMV5 and JMC7 which can allow much more accumulation of shikimic acid in their culture supernatants. We also amplified the expression of aroG and tktA gene so as to improve the accumulation level. The assay of shikimic acid by spectrophotometric method indicated that knocked out mutants accumulated two times lower shikimic acid than wild types with an intact shA gene. The expression of aroG and tktA showed that optimization of shikimic acid accumulation can be achieved by combining gene knockout and over-expression approaches. Among the knocked out strains; namely JMV5 and JMC7, JMC7 accumulated two times more shikimic acid than the JMV5. It is believed that the high level of accumulation in JMC7 strain results from the deletion of aroL gene into the strain. The supplement of shikimic acid into culture medium elucidated that shiA gene was completely knocked out. The entire shikimic acid did not cross the membrane to enter the cell. Moreover, it was a significant reduction of shikimic acid flow from inside the cell to the culture medium into shiA gene knocked out mutant. These results suppose that shiA gene may act in both directions because its disruption prevented the flow of shikimic acid to culture medium, meaning that shiA gene did not improve the accumulation into culture medium. In this study, it was not possible to detect the in vivo shikimic acid due to too low accumulation.