Anti-angiogenesis gene therapy is considered a promising treatment for excessive vascularization. Mesenchymal stem cell (MSC)-based gene therapy may enhance the effect of anti-angiogenesis by maintaining a long therapeutic period in vivo. However, transduction efficiencies and transgene expression in MSC-based gene therapy should be improved. Here we report human placenta-derived MSC (HPMSC)-based gene therapy using a fiber-modified adenoviral vector carrying the kringle1-5 gene to maintain long-term survival and effectively suppress angiogenesis both in vitro and in vivo. HPMSCs infected by the adenoviral vector were transduced at high efficiency with a low multiplicity of infection, and the infected HPMSCs expressed exogenous kringle1-5 protein in vitro and in vivo. Infected HPMSCs were detected at 2 weeks in vivo by fluorescence imaging and immunohistochemistry of reporter gene expression. Importantly, the microvessel growth of aortic rings in vitro was inhibited by administration of infected HPMSCs expressing kringle1-5 protein (K1-5-HPMSCs) at day 6. In Matrigel plugs combined with K1-5-HPMSCs, microvessel density was decreased as detected by immunohistochemistry and blood flow was decreased as detected by the power Doppler contrast enhanced at day 14. The fiber-modified adenovirus is an effective gene vector for HPMSC-based gene therapy, which may be a promising strategy for cancer anti-angiogenesis.