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DNA methylation at the five position of cytosine is well recognized as an important epigenetic modification in human health and disease. Recent evidences demonstrated that 5-methylcytosine (5-mC) by the TET family of enzymes can be converted to 5-hydroxymethylcytosine (5-hmC). Here, we use an ultrasensitive and accurate isotope-based LC–MS/MS method to precisely determine the levels of 5-hmC and 5-mC in colorectal cancer and the C-26 colon adenocarcinoma cell line.Our data showed that 5-hmC content is significantly reduced (approximately sixfold) in colorectal cancer as compared with adjacent normal tissue. Similarly, the ratio of 5-hmC to 5-mC dropped from 0.054 ± 0.005 in normal tissues, to 0.011 ± 0.002 in cancer.The analysis of 5-hmC levels and the ratio of 5-hmC:5-mC during tumor progression might provide insight into the role of this modification in cellular immortalization and transformation.