The tumor microenvironment composites a mixture of immune lymphoid cells, myeloid cells, stromal cells with complex cytokines, as well as numerous lymphovascular vessels. Colorectal cancer (CRC) is a common malignancy and one of the leading causes of tumor-related death in the United States and worldwide. The immune status in the tumor microenvironment contributes to the survival of a patient with CRC. Regulatory T cells (Tregs) are considered a key factor in immune escape and immunotherapy failure among cancer patients. The transcription factor forkhead box P3 (FOXP3) is a crucial intracellular marker and also a key developmental and functional factor for CD4+CD25+ Tregs. Tregs are correlated with survival in various human neoplasms, and elevated proportions of Tregs are usually associated with unfavorable clinical outcomes. However, the role of Tregs in CRC remains controversial. High densities of tumor-infiltrating Tregs in CRC patients are reported to be correlated with worse or better outcomes. And Tregs may not be predictive of prognosis after resection of the primary tumor. The exact explanations for these discordant results remain unclear. The heterogeneous instincts of cell phenotype, gene expression, and functional activities of Tregs may partly contribute this contrasting result. Furthermore, the lack of a robust marker for identifying Tregs or due to the different techniques applied is also account. The Treg-specific demethylated region (TSDR) was recently reported to be a specific epigenetic marker for natural Tregs (nTregs), which can stably express FOXP3. The FOXP3-TSDR demethylation assay may be an promising technique for CRC-related nTregs studies.