Adult hemophilia B mice were treated with identical AAV vectors, encoding either the FIX CB 2679d-GT or R338L-Padua variants, at the same dose. Evaluation of treated animals demonstrated nearly identical circulating levels of FIX protein, liver vector copy number, and FIX mRNA levels. However, the new CB 2679d/ISU304 transgene, which is designed to have improved FIX functions such as catalytic activity, affinity for activated FVIII, and resistance to antithrombin inhibition, outperformed the R338L-Padua by providing rapid and robust hemostatic correction. Thus, the FIX variant CB 2679d-GT could improve tolerability of the vector in future gene therapy trials.