The purpose of this study was to determine the aberrant expression of the long noncoding RNA (lncRNA) colon cancer-associated transcript 1 (CCAT1) in 5-fluorouracil-resistant colonic neoplasm cells and to elucidate its effects on the 5-fluorouracil sensitivity of human colonic neoplasm cells. The aberrant expression of lncRNAs in normal tissues and colonic neoplasm tissues was detected by microarray assay. qRT-PCR analysis was performed to assess CCAT1 expression levels in colonic neoplasm cell lines and corresponding normal tissues. After constructing the 5-FU-resistant cell lines and validating the resistance by measuring the IC50 value, the CCAT1 expression levels in parental and artificially resistant cell lines were determined by qRT-PCR. Transfection was used to modulate the expression of CCAT1. Cell proliferation and apoptosis were then detected by CCK-8 and flow cytometry, respectively. CCAT1 in colon cancer tissues was higher than that in noncancer tissues, and the levels of CCAT1 in HCT 116, SW1417, HT-29, and KM12 cell lines were higher than those in the human normal colon epithelial NCM460 cell line. Moreover, the expression levels of CCAT1 were high in HCT 116/5-FU and HT-29/5-FU cell lines, whose apoptosis rates induced by 5-FU were lower than those in corresponding parental cells. The results of qRT-PCR and CCK-8 assay showed that enhancement of lncRNA CCAT1 expression levels in HCT 116 and HT-29 cell lines increased their IC50 of 5-FU and decreased their apoptosis rates. Meanwhile, siRNA-CCAT1 effectively inhibited the expression of CCAT1 and enhanced the 5-FU-sensitivity of HCT 116/5-FU and HT-29/5-FU, in which apoptosis rates were increased at the same time. Downregulation of CCAT1 effectively reversed the resistance of HCT 116/5-FU and HT-29/5-FU cells to 5-FU chemotherapeutic, opening a new avenue in colon cancer therapy.