Fibroblast growth factor-21 (FGF-21) is a potential cytokine for type II diabetes mellitus. This study aimed to optimize recombinant human FGF-21 (rhFGF-21) production in Escherichia coli BL21 (DE3) employing high cell density fermentation at a 200-L scale and pilot-scale purification. FGF-21 was eventually expressed in E. coli BL21 (DE3) using human FGF-21 synthetic DNA sequence via the introduction of vector pET-3c; the product is used as seed strain during the fermentation of rhFGF-21. Fermentation of rhFGF-21 was performed in a 30-L and 200-L fermenters. rhFGF-21 was primarily expressed in the form of inclusion bodies after IPTG induction. At the 200-L scale, the bacterial production and expression levels of rhFGF-21 were 38.8 +/- 0.6g/L and 30.9 +/- 0.7%, respectively. Additionally, the high purification (98%) of rhFGF-21 was tested with HPLC analysis and reducing & non-reducing SDS-PAGE analysis. The final yield of purified rhFGF-21 was 71.1 +/- 13.9mg/L. The activity of rhFGF-21 stock solution reached at 68.67 +/- 8.74IU/mg. Blood glucose controlling and insulin sensitization were improved with treatment of rhFGF-21 in type II diabetic ob/ob mice. Our results showed that the relatively stable and time-saving pilot-scale production process was successfully established, providing an efficient and cost-effective strategy for large-scale and industrial production of rhFGF-21.