Correlations between drug resistance and glycosylation changes have been analyzed intensively in the field of tumor biology. The present study was aimed to investigate the glycan and glycogene alterations involved in oxaliplatin resistance in human colon cancer cells. Using the lectin microarray for glycan composition and FITC-lectin binding for glycan profiling, we found that polylactosamine-type N-glycans were significantly increased in oxaliplatin-resistant SW620R cells. Using real-time PCR for quantification of glycogenes, we targeted β-1,3-N-acetylglucosaminyltransferase 8 (β3GnT8), which was overexpressed in SW620R cells. Using an RNA interference strategy, we revealed that the silencing of β3GnT8 in SW620R cells resulted in increased chemosensitivity to oxaliplatin. Conversely, the engineered overexpression of β3GnT8 in SW620 cells enhanced resistance to oxaliplatin. Further data revealed that manipulation of β3GnT8 was able to modify polylactosamine chains on integrin β1 and to regulate the integrin β1 downstream signaling pathway. These results revealed that β3GnT8 may play a key role in the development of oxaliplatin resistance in colon cancer cells possibly through the alteration of the glycosylation of integrin β1. These findings may be valuable for overcoming drug resistance in colon cancer.