CD133, a five-transmembrane molecule, has been found on many types of cancers and determined to be a cancer stem cell biomarker. In this study a functional anti-human CD133 MAb 6B6 was obtained, and the specificity of this MAb was verified by flow cytometry. This MAb effectively recognized the CD133 molecule expressed on a series of malignant cell lines. Immunohistochemistry staining showed the CD133 was expressed on colorectal tumor tissue. Furthermore, we demonstrated that MAb 6B6 could inhibit the proliferation of Caco-2 cells that were derived from a human colorectal carcinoma. This functional anti-human CD133 MAb provides a valuable tool for further study of biological functions of cancer stem cell that expressed CD133.