This study found that, when compared with control cells in vitro, CD133(+)-shHOTAIR exhibited the decreased HOTAIR expression, suppressed cellular proliferation, migration, invasion, colony-forming, and inhibited the Vimentin expression with increased E-cadherin expression. In particular, the down-regulation of the HOTAIR expression in CD133(+)CSCs markedly attenuated the tumor growth and lung metastasis in xenograft nude mice. Taken together, this study found that down-regulating the HOTAIR expression in CD133(+)CSCs could serve as a potential anti-cancer regimen to inhibit the invasiveness and metastasis of CRC CSCs.