The vascular endothelial growth factor (VEGF) is an essential factor to pathologic angiogenesis. Disruption of VEGF/VEGF receptor interaction in cancer patients inhibits the development of new and pre-existing tumor blood vessels. Consequently, VEGF becomes an important therapeutic target for handling solid tumors. In this work, human VEGF was produced in the culture supernatant of SiHa cells transduced with a replication-defective adenoviral vector (pAdhVEGF(121)) encoding this molecule. The 35 kDa VEGF(121) homodimer was obtained from clarified culture media as a glycosylated protein. VEGF(121) expression levels were strictly dependent on the adenoviral viral load used. VEGF(121) was produced with purity over 98% after a single step chromatography by immobilized metal affinity chromatography. Additionally, VEGF(121) binds Bevacizumab antibody with a K-D of 7 nM. Biological characterization by mitogenic assay in HUVEC and ECV-304 cells showed that VEGF(121) stimulates cell proliferation in a dose-dependent manner in both cells. Finally, the neovascularization activity of VEGF(121) was demonstrated by vascular permeability assays in matrigel plug-bearing mice, showing significantly increased vasculature leakage after treatment with VEGF(121). Consequently, transduction of SiHa cells with adenovirus is a suitable alternative for manufacture heterologous proteins of therapeutic interest.