Abstract Aims To investigate the immune microenvironment modulation of iNKT cells for recognition and elimination of colorectal metastasis-initiating cell (MIC). Methods Different subtypes of human colorectal cancer cells and iNKT cells were analyzed by BD LSRFortessa™, TMA, and immunofluorescence(n=138). Microfluidic circulating tumor cell enrichment, Ex vivo 3D co-culture, Quansys Multiplex array, survival data analysis, and Elisa array were performed. Results There is a subtype of colorectal cancer stem cell referred as a metastasis-initiating cell (MIC), which do not express upregulated mesenchymal genes but express elevated levels of CD36 (the fatty acid receptor) and other lipid metabolism genes. Intriguingly, those cells overexpress NKG2DL, Various immune cells express NKG2D receptors, including natural killer (NK) cells, CD8+ T cells, and human iNKT cells. Concerning the NKT cells, there is an increase of NKG2D expression on liver resident iNKT cells (P < 0.05). We further observed that hepatic iNKT cells located at the invasive margin had been activated as shown by increased IFN-γ, TNF-α, granzyme B and Ki-67 expression. Furthermore, transmembrane CD1d density and intra/tmCXCL16 expression were also elevated in CD36+colorectal cancer stem cells, those cells showed significantly elevated α-GalCer: CD1d complex formation than other cancer stem cells in an ex vivo iNKT cells activation assay. CD36+cancer stem cells show susceptibility to iNKT cells induced cytotoxicity.