Hemophilia B is a hereditary bleeding disorder characterized by a deficiency in coagulation factor IX (F.IX). Directing point mutations in F.IX that decrease retention in tissue and increase specific activity can improve gene and protein-based methods of treatment for the disease. Translation of these mutations to canine F.IX offers an ideal system for studying the safety and efficacy of these methods in a canine model. This study shows the successful production and purification of recombinant canine F.IX, as well as improved biological activity of the variant. Analysis of protein from stable cell lines shows that a mutation R338A improves specific activity for the variant 3-10 fold. Furthermore, cells transduced with viral vectors encoding canine F.IX reproduced this finding. These results can be used to improve skeletal muscle-directed gene therapy for Hemophilia B or protein therapy by providing highly active recombinant protein for injection.