Shikimic acid (3, 4, 5-trihydroxy cyclohexene carboxylic acid; SA) is a key intermediate for the synthesis of the oseltamivir phosphate (Tamiflu), and it has several innovative and multifunctional roles in dermocosmetic preparations, anti-enzymatic activity, and as an exfoliating agent. In this study, production SA by using E. coli was carried out. The process of producing SA was optimized by determining effect of various nutritional and physical parameters. The maximum SA production titer (33.44 g/L) and yield (43.1%) were achieved, which was the highest value ever reported from glycerol. These results suggest that utilization of not only currently used glucose but glycerol as an alternative carbon source is much effective for the production of SA in E. coli. Moreover, these data would lay the foundation for future research on the utilization of this strain from glycerol and to achieve higher SA production efficiency.