MicroRNAs are small RNAs that regulate gene expression at the post-transcriptional level, which always showed abnormally expressed in cancer.We found that miR-128 is abnormally expressed in colon cancer, but the role of miR-128 in colon cancer still keeps indistinct.Compared with absent tissue, the miR-128 level decreased in colon cancer clinical samples by qPCR assay.Overexpression of miR-128 could inhibit cell growth and decreased chemosensitivity to methotrexate in HCT116.Flow cytometry assay showed miR-128 induced cell cycle arrest by decreasing S phase.We found that prohibitin (PHB) is one of the targets of miR-128, Western blotting also showed that miR-128 could decrease PHB protein expression level, increase P53 and P21 protein levels.We also found that miR-128 could inhibit migration in colon cancer cell line, the mechanism need more intensively work.Our work showed that miR-128 could induce cell cycle arrest by inhibiting S phase in colon cancer cell line, the mechanism might accomplished by targeting PHB-P53-P21 pathway.