A major problem associated with current cancer therapeutics is the eventual recurrence even after the elimination of bulk of the cancer cells.Recent studies on cancer stem cells may provide a potential solution to this problem.We report here the identification of internalizing human single-chain antibodies(scFv) targeting Glioblastoma Multiform(GBM)tumor sphere cells.A large naive phage display antibody library was constructed and selected on the glycosylation-dependent CD 133 epitope-positive subpopulation of GBM cells grown as tumor spheres and identified internalizing scFvs that target tumor sphere cells broadly, as well as scFvs that target the CD133-positive subpopulation.These scFvs were found to be efficiently internalized by GBM tumor sphere cells.One scFv GC4 inhibited self-renewal of GBM tumor sphere cells in vitro.We have further developed a full-length human IgG1 based on this scFv, and found that it potently inhibits proliferation of GBM tumor sphere cells and GBM cells grown in regular nonselective medium.Plus, we developed a nanoparticle formulation modified with tumor-targeting GC4 single-chain antibody fragment (scFv) for systemic delivery of small interfering RNA (siRNA) and microRNA (miRNA).Taken together, these results show that internalizing human scFvs targeting tumor cells can be readily identified from a phage antibody display library, which could be useful for further development of novel therapies.