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S100 calcium binding protein A4 (S100A4) promotes extracellular signal transduction, intercellular adhesion, motility and mobility. Different extracts from Coix lachryma-jobi have been used for the treatment of various types of cancer in Asia. In our previous study, the polysaccharide fraction extact, CP1, induced cell apoptosis of non-small cell lung cancer cells. In the current study, CP1 inhibited migration and invasion of A549 cells in a scratch wound healing assay and matrigel invasion assay, respectively. Furthermore, reverse transcription-polymerase chain reaction and western blotting demonstrated that CP1 downregulated the gene and protein expression levels of SI00A4. In silico docking analysis demonstrated that polysaccharides may not interfere with dimerization, whereas, the affinity of polysaccharides for an S100A4-NMIIA pocket was margnially greater than at the dimerization sites. Thus, CP1 inhibited A549 cell migration and invasion potentially via downregulation of S100A4, and may also interact with the binding site of S100A4-NMIIA, which indicated that CP1 has potential as an alternative cancer chemotherapeutic by targeting S100A4.