One major reason for the failure of advanced colorectal cancer (CRC) treatment is the occurrence of chemoresistance to fluoropyrimidine (FU)-based chemotherapy. Long non-coding RNA HOTAIR has been considered as a pro-oncogene in multiple cancers. However, the precise functional mechanism of HOTAIR in chemoresistance is not well known. In this study, we investigated the biological and clinical role of HOTAIR in 5FU resistance in CRC. Our results showed that HOTAIR negatively regulated miR-218 expression in CRC through an EZH2-targeting miR-218-2 promoter regulatory axis. HOTAIR knockdown dramatically inhibited cell viability and induced G1-phase arrest by promoting miR-218 expression. VOPP1 was shown to be a functional target of miR-218, and the main downstream signaling, NF-κB, was inactivated by HOTAIR through the suppression of miR-218 expression. Additionally, HOTAIR knockdown partially reversed 5FU resistance through promoting miR-218 and inactivating NF-κB signaling. Furthermore, HOTAIR restrained 5FU-induced cytotoxicity on CRC cells through promotion of thymidylate synthase expression. More importantly, high HOTAIR expression was associated with poor response to 5FU treatment. In conclusion, we demonstrated that HOTAIR contributes to 5FU resistance through suppressing miR-218 and activating NF-κB signaling in CRC. Thus, HOTAIR may serve as a promising therapeutic target for CRC patients. Keywords: HOTAIR, colorectal cancer, 5-fluorouracil, NF-κB, thymidylate synthase, miR-218