Objective ·To investigate the composition of immune cells in tumor microenvironment of colorectal cancer (CRC), and examine the proportion, phenotype and effector function of natural killer (NK) cells in CRC. Methods ·Fresh tumor tissues, paired normal tissues adjacent to tumor, and peripheral blood samples in the same cohort were collected from CRC patients. Tissues were digested and prepared into single cell suspension. The major immune cell lineages were detected by flow cytometry. t-Distributed stochastic neighbor embedding (t-SNE) and statistical analysis were used to analyze the composition of immune cells in tumor microenvironment of CRC. To analyze the phenotype of NK cells, the expression levels of activation markers, including CD16, CD27, CD69, human leukocyte antigen-DR (HLA-DR), and T cell immunoglobulin domain and mucin domain-3 (TIM-3), were detected by flow cytometry. NK cell subsets: CD38^lowNK cells and CD38^highNK cells were also examined by flow cytometry. To assess the effector function of NK cells, they were stimulated with cell stimulation cocktail and the expression levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were measured by flow cytometry. Results ·Twenty-five pairs of fresh tumor tissues and normal tissues adjacent to tumor, and 15 peripheral blood samples in the same cohort were collected from CRC patients. The tumor microenvironment of CRC included diverse immune cell types, including T cells, B cells, NK cells and myeloid cells. The proportions of T cells (P=0.000) and myeloid cells (P=0.026) in tumor tissues were significantly higher than those in normal tissues. By contrast, the proportion of NK cells (P=0.007) in tumor was significantly reduced. The proportion of B cells was comparable between tumor and normal tissues. Compared to normal tissues, NK cells in tumor tissues expressed significantly lower CD27 (P=0.000) and CD69 (P=0.001), while the expression levels of CD16 (P=0.008), HLA-DR (P=0.000) and TIM-3 (P=0.024) were significantly elevated. The results indicated that NK cells in CRC tumor exhibited a phenotype of late activation and exhaustion. According to the expression level of CD38, NK cells could be divided into two subsets, CD38^highNK cells and CD38^lowNK cells. The proportion of CD38^highNK cells (P=0.003) in tumor tissues was significantly lower than that in normal tissues, while the proportion of CD38^lowNK cells was unaffected. Compared to CD38^lowNK cells, CD38^highNK cells expressed higher CD27, meanwhile significantly less CD16, NKp46, CD57, CD94, HLA-DR and CD158a (P=0.000). These results suggested that CD38^highNK cells were at early differentiation state. The secretion of cytokines IFN-γ (P=0.032), TNF-α (P=0.042), and GM-CSF (P=0.019) by tumor-infiltrated NK cells was significantly decreased compared to that in normal tissues. The results showed that the function of tumor-infiltrated NK cells was impaired. Conclusion ·Together, these data suggest that NK cell compartment is disrupted in tumor tissues of CRC, leading to the impaired anti-tumor immunity mediated by NK cells.