A number of reports have described the production of polysialic acid (PSA), focusing on the fermentation and purification of PSA. However, little work has been done to strengthen the synthetic pathway of PSA to improve PSA production. In this study, an efficient process for enhanced production of PSA using a recombinant Escherichia coli strain was developed. To improve the PSA production efficiency, the key enzymes of PSA synthetic pathway were overexpressed separately or in combination, including N-acetylneuraminate (Neu5Ac) 7-O(or 9-O)-acetyltransferase (NeuD), CMP-Neu5Ac synthetase (NeuA), and alpha-Neu5Ac alpha-2,8-sialyltransferase (NeuS). The PSA production was significantly improved by coexpression of NeuD and NeuA. In terms of the efficiency, NeuD was considered as the most important factor. Secondly, the competing pathway of intermediate Neu5Ac was blocked by nanA deletion. The efficient PSA-producing strain E. coli SA9 Delta nanA/pDB1S-DA was constructed, and 16.15 +/- 1.45 g/L PSA was obtained in the fed-batch culture. The production of PSA by engineered strain was increased by 85 % compared to the original strain. These results provide evidence for improvement of PSA production by regulation of the PSA biosynthetic pathway. The high productivity of our process should make it a promising cost-effective resource for PSA.