Stromal cell-derived factor-1 (SDF-1)/chemokine (CXC motif) ligand 12 (CXCL12) is involved in the process of tumor progression. Sulfated K5 polysaccharides have shown anti-cancer activity by acting on multiple targets, though it remains unclear whether sulfated K5 polysaccharides would disrupt SDF-1/CXCL12-stimulated cancer biology. This study aimed to investigate the effects of sulfated K5 polysaccharides on cell growth, adhesion in murine B16 melanoma cells and the underlying mechanism by targeting SDF-1/CXCL12. Results indicated that K5-NS,OS inhibited the proliferation of B16 melanoma cells, induced the cell cycle arrest mainly at the G0/G1 phase, and suppressed cancer cell proliferation or adhesion induced by SDF-1/CXCL12. It was possible that K5-NS,OS appeared to interact with CXCL12 and block the subsequent biological functions. This work suggests that the existence of O- and N-sulfate groups is more effective in targeting CXCL12 and exhibiting anti-cancer activity.