Cancer is a large and increasing burden on global healthcare systems. Current treatment options for most cancers are considered effective if the cancer is detected early. The life expectancy severely decreases with late-stage diagnosis and with cancer progression, unfortunately, most cancers are diagnosed at advanced stages (Pulumati et al., 2023). Hence the need to develop potential biomarkers of metastasis that could be used as diagnostic tools. The primary aim of this research is to identify global glycan changes associated with the effects of N-acetylglucosaminyltransferase (GCNT3) knockdown in human adenocarcinoma cells. GCNT3 has been characterised as a pro-metastatic gene (Lange et al., 2022), thus, identifying the changes that occur within the glycan profile associated with GCNT3 expression as potential biomarkers. A comprehensive glycan analysis, using Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC ESI-MS/MS), of three adenocarcinoma cell lines HT29, PACA5061, GC5023 each having three genotype variants, 'wild type', 'GCNT3 knockdown' and 'negative control', examined the changing glycan profiles based on GCNT3 expression. Altered GCNT3 expression significantly changed the O-glycan profiles of all adenocarcinomas. GCNT3 knockdown resulted in a decrease of core 2 glycan structures with a corresponding increase in core 1 structures with all adenocarcinomas having a sialylated Lewis structure significantly more abundant in GCNT3 variant compared to the GCNT3 knockdown variant. GCNT3 expression had a varied effect on glycan abundance within the N-glycan profile having significant changes but no commonality between cell lines. The effect of GCNT3 expression highlights many significantly different sialylated Lewis glycans which could be candidates for potential biomarkers of metastasis and areas of future research.