Aberrant glycosylation is known to be associated with cancer chemoresistance. β-1,3-N-acetyl-glucosaminyltransferase (β3GnT)8, which synthesizes polylactosamine on β1-6 branched N-glycans, is dramatically upregulated in colorectal cancer (CRC). 5-Fluorouracil (5-FU) resistance remains a major obstacle to the chemotherapy of CRC. However, little is known with regard to the correlation between 5‑FU resistance and the expression of β3GnT8 in CRC. In this study, a 5-FU‑resistant cell line (SW620/5-FU) was generated, and 50% inhibition concentration (IC50) of 5-FU was determined by MTT assay. Flow cytometry and lectin blot analysis were performed to detect the alteration of polylactosamine structures. Quantitative RT-‑PCR and western blot analysis were used to identify and evaluate candidate genes involved in the synthesis of polylactosamine in SW620/5-FU cells. We found polylactosamine chains were significantly increased in SW620/5-FU cells. Inhibition of the biosynthesis of polylactosamine by 3'-azidothymidine (AZT) was able to reduce 5-FU tolerance. Further studies showed that β3GnT8 expression was also upregulated in 5-FU‑resistant cancer cells, and knockdown of β3GnT8 by RNA interference reversed 5-FU resistance through, at least partly, by suppressing the formation of polylactosamine. In conclusion, the alteration of β3GnT8 in CRC cells correlates with tumor sensitivity to the chemotherapeutic drug and has significant implication for the development of new treatment strategies.