Increased expression of CD133 (Prominin-1), an important cancer stem cell-associated marker, has been observed in the cancer stem cells of a variety of human and mouse cancers. However, no natural ligand of CD133 has yet been identified and little is known about its function. In the present study, LS-7 (amino acid sequence: LQNAPRS), a specific binding peptide targeting mouse CD133, was screened and identified for the first time by phage-displayed peptide library technology. The in vitro and in vivo affinity and specificity of LS-7 were determined, and MTT, adhesion, and migration assays were performed to evaluate the effects of LS-7 on the biological behaviors of cancer cells. To determine which signaling pathways are affected by LS-7, HMGB1, S-100A4, CXCR7, uPAR, AMFR, STAT3, and c-Met gene and protein expression were evaluated by RT-PCR and Western blot. Flow cytometry and immunofluorescence assays showed specific, high-affinity binding of the peptide to mCD133 in vitro. Confocal microscopy confirmed the co-localization of LS-7 positive cells and CD133-positive cells. Migration and wound-healing assays showed that LS-7 significantly inhibited the migration of colon and breast cancer cells in a concentration-dependent manner. In vivo experiments also confirmed the high specificity and affinity of LS-7 to mCD133. RT-PCR and Western blot showed that the expressions of only c-Met and STAT3 decreased obviously in colon and breast cancer cells exposed to LS-7. These findings may provide a novel tool for anti-motility and anti-metastasis strategies in cancer research and cancer stem cell therapy.