Adeno-associated virus 8 (AAV8), a new member of the AAV family isolated from nonhuman primates, is an attractive candidate for hepatic gene transfer applications because of 10- to 100-fold improved transduction efficiency in mouse liver models. Additionally, AAV8 has lesser frequency of pre-existing immunity in humans. These properties could solve some of the problems associated with AAV2 vectors. The benefits of AAV8 demonstrated in mouse models, however, have not been confirmed in larger animals. In this study, we evaluate the efficacy and safety of AAV2/8 vector in both naive and AAV2-pretreated hemophilia B dogs. Two naive hemophilia B dogs that received a single intraportal administration of AAV2/8 vector have achieved sustained expression of 10% and 26% of normal levels of canine factor IX (cFIX) for more than a year. In an AAV2-pretreated hemophilia B dog, cFIX expression increased from less than 1% to 16% of normal levels when treated with an AAV2/8 vector, and a high level of expression has lasted for more than 2 years. No significant liver toxicity or cFIX-specific antibodies have been detected in these animals. Studies here have demonstrated the safety and improved efficacy of AAV2/8 vector in large-animal models for liver-directed gene therapy.