The functions of the precursor H antigen for ABO blood group antigens are still not fully understood, particularly in cancer cells. In this study, we used hybridoma technology and NSY human colon cancer cells as an immunogen to generate a monoclonal antibody designated as MAb L9E10. The binding antigen of MAb L9E10 was identified as blood group (BG) H2 antigen using carbohydrate array and erythrocyte agglutination assays. In immunofluorescence study, we found that BG-H2 was expressed on the surfaces of both colon cancer stem cells and their differentiated progeny. In a functional study, we observed that MAb L9E10 inhibited tumor cell migration and invasion at a concentration of 10 mg/mL in vitro. This result suggests that MAb L9E10 could be used to study cancer biology, particularly cancer stem cell biology. In addition, it is potentially useful for studying gastric diseases caused by Helicobacter pylori bacteria, with attachment to human gastric epithelial cells mediated by blood group antigens Lewis b and H2. Finally, MAb L9E10 is an ideal biological reagent for identifying Bombay blood type in which erythrocytes have no BG-H2 antigen expression.