We constructed a biosynthetic pathway of isoprene production in Escherichia coli by introducing isoprene synthase (ispS) from Populus alba. 1-deoxy-d-xylulose 5-phosphate synthase (dxs), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (dxr) and isopentenyl diphosphate (IPP) isomerase (idi) were overexpressed to enhance the isoprene production. The isoprene production was improved 0.65, 0.16, and 1.22 fold over the recombinant BL21 (pET-30a-ispS), respectively, and idi was found to be a key regulating point for isoprene production. In order to optimize the production of isoprene in E. coli, we attempted to construct polycistronic operons based on pET-30a with genes dxs, dxr, and idi in various orders. The highest isoprene production yield of 2.727 mg g(-1) h(-1) (per dry weight) was achieved by E. coli transformed with pET-30a-dxs/dxr/idi. Interestingly, the gene order was found to be consistent with that of the metabolic pathway. This indicates that order of genes is a significant concern in metabolic engineering and a sequential expression pattern can be optimized according to the biosynthetic pathway for efficient product synthesis.