Mucopolysaccharidosis type II (MPSII; Hunter syndrome) is a lysosomal storage disorder caused by a deficiency in the enzyme iduronate 2-sulfatase. At present, the therapeutic approaches for MPSII are enzyme replacement therapy and bone marrow transplantation, although these therapies have some limitations. The availability of new AAV serotypes that display tissue-specific tropism and promote sustained expression of transgenes offers the possibility of AAV-mediated gene therapy for the systemic treatment of lysosomal diseases, including MPSII. We have characterized in detail the phenotype of iduronate-2-sulfatase-deficient mice, a model of human MPSII. These mice display a progressive accumulation of glycosaminoglycans in many organs and excessive excretion of these compounds in their urine. Furthermore, they develop skeleton deformities, particularly of the craniofacial bones, and alopecia, they perform poorly in open field tests, and they have a severely compromised walking pattern. In addition they present neuropathological defects. We have designed an efficient gene therapy approach for the treatment of these MPSII mice. AAV2/8TBG-IDS viral particles were administrated intravenously to adult MPSII mice. The plasma and tissue iduronate 2-sulfatase activities were completely restored in all of the treated mice. This rescue of the enzymatic activity resulted in the full clearance of the accumulated glycosaminoglycans in all of the tissues analyzed, the normalization of the glycosaminoglycan levels in the urine, and the correction of the skeleton malformations. Overall, our findings suggest that this in vivo gene transfer approach has potential for the systemic treatment of patients with Hunter syndrome.