The gut microbiome influences the response to immunotherapy in non-small cell lung cancer (NSCLC). This study investigated the impact of gut microbiota on immunotherapy effectiveness using clinical samples and animal models. The results showed that higher gut microbiota diversity was associated with better responses to immune checkpoint inhibitors (ICIs). Patients who responded to treatment (R) had increased levels of Faecalibacterium and short-chain fatty acids (SCFAs), including butyric acid, acetic acid, and hexanoic acid. Fecal microbiota transplantation (FMT) from R and nonresponders (NR) improved anticancer effects and reduced Ki-67 expression in tumors. However, FMT did not significantly affect PD-L1 expression. The diversity of gut microbiota correlated with a better prognosis in NSCLC patients, potentially mediated by SCFAs. The study suggests that gut microbiota diversity and SCFAs are related to immunotherapy efficacy, and FMT can enhance immunotherapy outcomes. The findings highlight the potential of FMT in improving immunotherapy for NSCLC patients. The study also indicates that gut microbiota composition and SCFAs may influence immunotherapy response, with Faecalibacterium showing a correlation with immunotherapy efficacy. Further research is needed to explore the mechanisms underlying the effects of gut microbiota on immunotherapy and to determine the optimal use of FMT in clinical settings. The study has limitations, including the use of a mouse model and a small sample size, which may affect the generalizability of the findings. Overall, the study provides evidence that gut microbiota and SCFAs play a significant role in the response to immunotherapy in NSCLC patients.The gut microbiome influences the response to immunotherapy in non-small cell lung cancer (NSCLC). This study investigated the impact of gut microbiota on immunotherapy effectiveness using clinical samples and animal models. The results showed that higher gut microbiota diversity was associated with better responses to immune checkpoint inhibitors (ICIs). Patients who responded to treatment (R) had increased levels of Faecalibacterium and short-chain fatty acids (SCFAs), including butyric acid, acetic acid, and hexanoic acid. Fecal microbiota transplantation (FMT) from R and nonresponders (NR) improved anticancer effects and reduced Ki-67 expression in tumors. However, FMT did not significantly affect PD-L1 expression. The diversity of gut microbiota correlated with a better prognosis in NSCLC patients, potentially mediated by SCFAs. The study suggests that gut microbiota diversity and SCFAs are related to immunotherapy efficacy, and FMT can enhance immunotherapy outcomes. The findings highlight the potential of FMT in improving immunotherapy for NSCLC patients. The study also indicates that gut microbiota composition and SCFAs may influence immunotherapy response, with Faecalibacterium showing a correlation with immunotherapy efficacy. Further research is needed to explore the mechanisms underlying the effects of gut microbiota on immunotherapy and to determine the optimal use of FMT in clinical settings. The study has limitations, including the use of a mouse model and a small sample size, which may affect the generalizability of the findings. Overall, the study provides evidence that gut microbiota and SCFAs play a significant role in the response to immunotherapy in NSCLC patients.