A fiber-modified adenovirus (rAd5F11B), loaded with the Kringle1-5 gene (rAd-K1-5) was used to infect human adipose tissue-derived mesenchymal stem cells (HAMSCs). At a multiplicity of infection of 20, the transfection efficiency in HAMSCs was 90% and the cell expansion and differentiation of infected HAMSCs were not significantly suppressed. HAMSCs infected with rAd-K1-5 expressed the exogenous Kringle1-5 protein, an angiogenic inhibitor, and conditioned media from HAMSCs expressing the Kringle1-5 protein blocked VEGF-induced neovascularization both in vitro and in vivo. rAd5F11B may therefore be a promising gene transfer vector in HAMSCs-based anti-angiogenic gene therapy because of its low toxicity and high transfection efficiency.