Objective To develop recombinant factor IX (FIX) variants with augmented clotting activity. Results We generated three new variants, FIX-YKALW, FIX-ALL and FIX-LLW, expressed in SK-Hep-1 cells and characterized in vitro and in vivo. FIX-YKALW showed the highest antigen expression level among the variants (2.17 mu g(-mL)), followed by FIX-LLW (1.5 mu g(-mL)) and FIX-ALL (0.9 mu g(-mL)). The expression level of FIX variants was two-five fold lower than FIX-wild-type (FIX-WT) (4.37 mu g(-mL)). However, the biological activities of FIX variants were 15-31 times greater than FIX-WT in the chromogenic assay. Moreover, the new variants FIX-YKALW, FIX-LLW and FIX-ALL also presented higher specific activity than FIX-WT (17, 20 and 29-fold higher, respectively). FIX variants demonstrated a better clotting time than FIX-WT. In hemophilia B mice, we observed that FIX-YKALW promoted hemostatic protection. Conclusion We have developed three improved FIX proteins with potential for use in protein replacement therapy for hemophilia B.