5-Fluorouracil (5-FU)-based chemotherapy has always been the first-line treatment of colorectal cancer (CRC). However, the occurrence of clinical 5-FU resistance is a major reason for CRC therapy failure. This study intended to explore the possible role of long non-coding RNA HOTAIRM1 (HOTAIRM1) in the pathogenesis of 5-FU resistant CRC and its underlying mechanism. Our data showed that HOTAIRM1 was downregulated in CRC tissues and cell lines (HCT116 and SW480), and even lower in 5-FU resistant CRC tissues and cell lines (HCT116/5-FU and SW480/5-FU). In vitro, effects of HOTAIRM1 dysregulation in 5-FU resistant CRC cells were investigated and its overexpression could reduce cell viability, invasion, migration, and multi-drug resistance as evidenced by MTT assay, Transwell assay, epithelial-mesenchymal transition (EMT), and western blot analyzing expression of drug-resistant genes MRP1 and MDR1, respectively. Mechanically, dual-luciferase reporter assay and RNA immunoprecipitation (RIP) identified that HOTAIRM1 and B-cell translocation gene 3 (BTG3) were target genes of miR-17-5p. Moreover, miR-17-5p was upregulated and BTG3 was downregulated in HCT116/5-FU and SW480/5-FU cells. Silencing of miR-17-5p showed suppressive role on cell viability, invasion, migration, and multi-drug resistance in HCT116/5-FU and SW480/5-FU cells, which could be abolished by HOTAIRM1 knockdown. Similarly, ectopic expression of miR-17-5p reversed BTG3-mediated inhibition on cell viability, invasion, migration, and multi-drug resistance. In vivo, the tumorigenesis of HCT116/5-FU cells when highly expressed HOTAIRM1 by lentivirus infection was inhibited through downregulating miR-17-5p and upregulating BTG3. In conclusion, HOTAIRM1 might act as a tumor-suppressor in 5-FU resistant CRC cells in vitro and in vivo through downregulating miR-17-5p/BTG3 pathway and inhibiting multi-drug resistance.