A high saturated fat diet induces fatty liver in mice. The fatty liver is triggered initially by an increase in PPARγ2 protein in the liver, which leads to increased expression of lipogenic genes. Inactivation of PPARγ2 may improve fatty liver induced by a high saturated fat diet. RNA interference of PPARγ2 should be a powerful tool to elucidate the role of PPARγ2 in the development of fatty liver. Here, we describe our method for constructing the adenovirus vector containing short hairpin RNA targeted for PPARγ2. Adenovirus vector-mediated RNA interference for PPARγ2 should be useful for clarifying the biological role of the PPARγ2 pathway in various tissues and for therapeutic application in a variety of diseases.