Simple SummaryIn recent years, growing evidence has suggested that the gut microbiome can significantly influence antitumor immunity, both within and outside the gastrointestinal tract, thereby affecting the efficacy of immune checkpoint inhibitors (ICIs. A link between microbiota composition and response to ICIs has been reported in both mouse and human studies. Gut microbial features depend on a delicate balance of tolerance for commensal microbiota and defense against various potentially pathogenic microbiota orchestrated by host immune system. Type 3 innate lymphoid cells (ILC3s) are a group of tissue-resident innate lymphocytes related to host immune cell–microbiome interactions. They orchestrate immunity, inflammation and tolerance in the intestines and any alterations in their functions can cause gut inflammation, colon cancer and immunotherapy resistance.AbstractType 3 innate lymphoid cells (ILC3s) are primarily tissue-resident cells strategically localized at the intestinal barrier that exhibit the fast-acting responsiveness of classic innate immune cells. Populations of these lymphocytes depend on the transcription factor RAR-related orphan receptor and play a key role in maintaining intestinal homeostasis, keeping host–microbial mutualism in check. Current evidence has indicated a bidirectional relationship between microbiota and ILC3s. While ILC3 function and maintenance in the gut are influenced by commensal microbiota, ILC3s themselves can control immune responses to intestinal microbiota by providing host defense against extracellular bacteria, helping to maintain a diverse microbiota and inducing immune tolerance for commensal bacteria. Thus, ILC3s have been linked to host–microbiota interactions and the loss of their normal activity promotes dysbiosis, chronic inflammation and colon cancer. Furthermore, recent evidence has suggested that a healthy dialog between ILC3s and gut microbes is necessary to support antitumor immunity and response to immune checkpoint inhibitor (ICI) therapy. In this review, we summarize the functional interactions occurring between microbiota and ILC3s in homeostasis, providing an overview of the molecular mechanisms orchestrating these interactions. We focus on how alterations in this interplay promote gut inflammation, colorectal cancer and resistance to therapies with immune check point inhibitors.