Human CD133 (prominin-1), a cell surface glycoprotein, is used as a marker of hematopoietic and neural stem cells. Antibodies that recognize a glycosylation-dependent CD133 epitope have been extensively used for enrichment of tumor initiating cells in a variety of cancers. These currently available antibodies are restricted for use in only a subset of biological assays. We have generated a novel anti-human CD133 monoclonal antibody, using a recombinant protein consisting of highly immunogenic amino acid residues selected from the native CD133 protein as an immunogen. The antibody (identified as clone 7) specifically recognizes the CD133 protein in a variety of immunological applications including Western blot, immunofluorescence, flow cytometry and immunohistochemistry. Further, clone 7 specifically recognizes an unmodified CD133 extracellular domain, and not its glycosylated epitope. In conclusion, the specificity and usefulness in a wide range of applications suggest that clone 7 could be a valuable tool to identify CD133 positive cells as well as to target them for therapy.