Fibroblast growth factor-21(FGF21), which was discovered in 2000 as a non-insulin-dependent cytokine to regulate blood sugar, is expected to become a candidate for anti-diabetes drugs. However, the biological activity of FGF21 was influenced by its weak instability and short half-life in vivo. In this study, to improve the stability of FGF21 in vivo, we added two arginines to the C-terminus of FGF21, and named as FGF21-2A. After being calculated by the software pI/Mw, the pI of FGF21-2A reached to 5.84 from 5.43. Then we expressed and purified the mutant protein for the examination of stability and bioactivity. When we used Bandscan5.0 to analyze the supernatant of Rosetta(DE3) lysates after induction, the protein expression levels of FGF21-2A increased by 10.6%. The in vivo half-life time of FGF21-2A was significantly prolonged in pharmacokinetics experiment. And, compared to the wild-type FGF21, FGF21-2A not only had a better capacity of stimulating glucose uptake in 3T3-L1 cells in vitro and stimulating glycogen synthesis of the model mice in vivo, but also provided a significantly long lasting effect on reducing blood glucose in the type Ⅰ and type Ⅱ diabetic animals. Moreover，the results of the real-time PCR showed that, after long term treatment, FGF21-2A could increase the mRNA expression of GLUT1 and GK and reduce the mRNA expression of G6P, which suggests FGF21-2A has the same mechanism of glucose regulation with the wild-type FGF21. In conclusion, modifying FGF21 with arginines increases the protein stability, and enhances the protein bioactivity, which lays the foundation for development of drugs targeting diabetes mellitus.