Musashi1 (Msi1), a member of the RNA-binding protein (RBP) family, is highly expressed in neural progenitor or stem cells for the maintenance of stemness as well as in various cancers. Emerging studies have demonstrated that it regulates cell processes by translational activation or suppresses specifically bound mRNA. In the present study, we initially reported remarkably increased expression of Msi1 in colon cancer tissues compared with adjacent non-tumor tissues. Knockdown of Msi1 significantly suppressed the proliferation, colony formation, tumorsphere formation and the progression of implanted colon cancers, and induced cell cycle attest at G0/G1 phase, along with the upregulated expression of p21(cip1). Reporter assays using a chimeric mRNA that combined luciferase and the 3'-UTR of p21(cip1) revealed that Msi1 decreased the reporter activity through the specific motif. Thus, the current results suggested that downregulation of Msi1 could inhibit the growth of colon cancers and Msi1 may be a promising therapeutic target molecule for human colon cancers.