Epigenetic alterations, such as CpG islands methylation and histone modifications, are recognized key characteristics of cancer. Glycogenes are a group of genes which epigenetic status was found to be changed in several tumors. In this study, we determined promoter methylation status of the glycogene beta-1,4-galactosyltransferase 1 (B4GALT1) in colorectal cancer patients. Methylation status of B4GALT1 was assessed in 130 colorectal adenocarcinomas, 13 adenomas, and in paired normal tissue using quantitative methylation specific PCR (QMSP). B4GALT1 mRNA expression was evaluated in methylated/unmethylated tumor and normal specimens. We also investigated microsatellite stability and microsatellite instability status and KRAS/BRAF mutations. Discriminatory power of QMSP was assessed by receiving operating curve (ROC) analysis on a training set of 24 colorectal cancers and paired mucosa. The area under the ROC curve (AUC) was 0.737 (95% confidence interval [CI]:0.591–0.881, P = 0.005) with an optimal cutoff value of 2.07 yielding a 54% sensitivity (95% CI: 35.1%–72.1%) and a specificity of 91.7% (95% CI: 74.1%–97.7%). These results were confirmed in an independent validation set where B4GALT1 methylation was detected in 52/106 patients. An inverse correlation was observed between methylation and B4GALT1 mRNA expression levels (r = −0.482, P = 0.037). Significant differences in methylation levels and frequencies was demonstrated in invasive lesions as compared with normal mucosa (P = 0.0001) and in carcinoma samples as compared with adenoma (P = 0.009). B4GALT1 methylation is a frequent and specific event in colorectal cancer and correlates with downregulation of mRNA expression. These results suggest that the glycogene B4GALT1 represent a valuable candidate biomarker of invasive phenotype of colorectal cancer. © 2012 Wiley Periodicals, Inc.