SIRT1, a NAD(+) dependent class III deacetylase, takes part in many important biological processes. Previous studies show that SIRT1 is overexpressed in some cancers and plays an essential role in tumorigenesis. However, the association between SIRT1 and colorectal cancer (CRC) is still unclear. We found that many CRC specimens had strong SIRT1 expression, which had an obvious correlation with poor prognosis of CRC patients. Meanwhile, SIRT1 expression had a co-localization with CD133, a current universal marker to characterize colorectal cancer stem cells (CSCs). In vitro studies also revealed that SIRT1 was overexpressed in colorectal CSC-like cells. Moreover, SIRT1 deficiency decreased percentage of CD133(+) cells, attenuated the abilities of colony and sphere formation, and inhibited tumorigenicity in vivo in CRC cells. Further study demonstrated that the expressions of several stemness-associated genes, including Oct4, Nanog, Cripto, Tert and Lin28, were reduced by SIRT1 knockdown in CRC cells. Taken together, our findings suggest that SIRT1 plays a crucial role in keeping the characteristics of CSCs cells. SIRT1 is a potential independent prognostic factor of CRC patients after tumor resection with curative intent, and will contribute to providing a promising new approach to target at CSCs in CRC treatment.