This study investigates the expression and clinical significance of N-acetylglucosaminyltransferase V (MGAT5) in colon adenocarcinoma (COAD) using bioinformatics methods. The mRNA and protein expression of MGAT5 in COAD tissues and the expression of MGAT5 in various cell lines of COAD were analyzed using the databases. The relationship between MGAT5 expression and clinical pathological features of COAD was analyzed by UALCAN. The relationship between MGAT5 and cellular immune infiltration in COAD was analyzed in TIMER database; the relationship between MGAT5 and prognosis of COAD patients was analyzed in GEPIA. The co-expression genes of MGAT5 in COAD were obtained on Linkedomics and imported into Metascape for pathway enrichment analysis. We predicted and constructed MGAT5 protein–protein interaction network in human samples using the STRING database. Compared with normal colon tissues, MGAT5 exhibited high expression in both mRNA and protein levels, and MGAT5 was also overexpressed in most COAD cell lines. There were significant differences in expression level of MGAT5 in different pathological stages of COAD. MGAT5 was positively correlated with the infiltration of CD8 + T cells, CD4 + T cells, macrophages, neutrophils and dendritic cells. The disease-free survival of MGAT5 low expression group was better than that of MGAT5 high expression group. The genes co-expressed with MGAT5 were predominantly implicated in the Notch signaling pathway and N-glycan biosynthesis processes. MGAT5 was significantly upregulated in COAD and correlated with poor prognosis and immune infiltration in COAD. This suggested that MGAT5 may serve as a valuable biomarker for clinical prognosis and a potential target for immunotherapy in COAD.