Background We have found that the gene expression of heparin sulfate d -glucosaminyl 6-O-sulfotransferase-2 (HS6ST2) is overexpressed in colorectal cancer in a previous study. Aims of this study are to evaluate the HS6ST2 expression in colorectal cancer tumor samples in relation to the clinicopathological features. Methods The mRNA expression levels of HS6ST2 were analyzed in various cancer cell lines using real-time RT-PCR. HS6ST2 protein expression was evaluated using immunohistochemistry. Tumor specimens were obtained from 103 colorectal cancer patients operated in Kinki University Hospital. Results High mRNA expression was observed in 2/15 (13%) of gastric cancer and 3/6 (50%) of colorectal cancer cell lines. In immunohistochemistry analysis, cytoplasm staining was found in cancer cells, not in normal colon cells, and positive staining of HS6ST2 expression was seen in 37 patients (36.3%). There were no relationships between clinicopathological features and HS6ST2 expression in cancer cells. Colon cancer patients with HS6ST2 expression tend to have worse overall survival (P = 0.097, tre log-rank test). Conclusion The cytoplasm expression of HS6ST2 is tumor-specific in colon tissues. HS6ST2 may contribute to be worse outcome.