The gut microbiome plays a crucial role in cancer immunotherapy, influencing immune responses and treatment efficacy. Preclinical and human studies have shown that specific gut microbial populations and metabolites can enhance the effectiveness of immunotherapy, particularly immune checkpoint inhibitors (ICIs). Key mechanisms include the activation of pattern recognition receptors, molecular mimicry, and modulation by microbe-derived metabolites such as short-chain fatty acids (SCFAs) and tryptophan metabolites. Microbial interventions, including fecal microbiota transplantation (FMT), probiotics, prebiotics, and dietary interventions, have shown promise in improving immunotherapy outcomes. However, challenges remain, such as inter-individual variability, lack of mechanistic understanding, and safety concerns. Future research should focus on large-scale and longitudinal studies, multidisciplinary collaboration, mechanistic investigations, and protocol standardization to optimize microbial interventions in cancer immunotherapy. Advances in sequencing technology and engineered bacteria offer new opportunities for enhancing the efficacy of immunotherapy.The gut microbiome plays a crucial role in cancer immunotherapy, influencing immune responses and treatment efficacy. Preclinical and human studies have shown that specific gut microbial populations and metabolites can enhance the effectiveness of immunotherapy, particularly immune checkpoint inhibitors (ICIs). Key mechanisms include the activation of pattern recognition receptors, molecular mimicry, and modulation by microbe-derived metabolites such as short-chain fatty acids (SCFAs) and tryptophan metabolites. Microbial interventions, including fecal microbiota transplantation (FMT), probiotics, prebiotics, and dietary interventions, have shown promise in improving immunotherapy outcomes. However, challenges remain, such as inter-individual variability, lack of mechanistic understanding, and safety concerns. Future research should focus on large-scale and longitudinal studies, multidisciplinary collaboration, mechanistic investigations, and protocol standardization to optimize microbial interventions in cancer immunotherapy. Advances in sequencing technology and engineered bacteria offer new opportunities for enhancing the efficacy of immunotherapy.