Peptide R54 (Pep R54; CXCR4 antagonist peptide 19) is an antagonistic polypeptide targeting CXCR4, known for its significant anticancer properties. It inhibits CXCR4-dependent cell migration, epithelial-mesenchymal transition, and the development of lung metastasis, offering better serum stability and higher CXCR4 affinity (IC50=20 nM) compared to the lead compound. Peptide R54 works synergistically with anti-PD-1 therapy to exhibit antitumor effects in vivo, enhancing granzyme activity and reducing Foxp3 cell infiltration. It is useful for research in colon cancer, ovarian cancer, and melanoma.