Background: Platinum-based neoadjuvant chemotherapy, such as gemcitabine/cisplatin (GC) or dose-dense methotrexate/vinblastine/doxorubicin/cisplatin (ddMVAC), followed by radical cystectomy (RC), represents the current standard of care for T2-T4aN0M0 muscle-invasive bladder cancer (MIBC). Regrettably, only 40% of patients who received neoadjuvant chemotherapy achieved a pathological complete response (pCR). Recently, several trials showed a promising efficacy of immune checkpoint inhibitors (ICIs) plus neoadjuvant chemotherapy. BGB-A317-2002 study indicated that the addition of tislelizumab to GC can increase the pCR rate to 54.5%, with a pathological downstaging rate of 77.3%. Nevertheless, a subset of patients failed to derive benefits from ICIs plus neoadjuvant chemotherapy. Methods: This study aimed to explore the impact and mechanisms of gut microbiota on the efficacy of neoadjuvant therapy through the analysis of fecal samples obtained from MIBC patients who received GC or GC plus ICIs. Here, we reported the impact of gut microbiota on treatment outcomes in patients who received GC. Results: A total of 54 patients who received neoadjuvant GC chemotherapy were enrolled. Among them, 23 (42.59%) exhibited a pathological response (responsive group), and 31 (57.41%) showed no response (non-responsive group; no imaging changes or disease progression). Metagenomic sequencing on fecal samples from patients before and after therapy showed that the Shannon index in species level (1.182 vs. 0.954, p=0.016) and abundance of Ruminococcus gnavus were significantly greater in the responsive group than non-responsive group. It suggested that responsive patients displayed higher species richness of gut microbiota. Conclusions: The efficacy of neoadjuvant GC chemotherapy in MIBC patients may be potentially influenced by the number of gut microbiota populations. Additionally, the potential application value of Ruminococcus gnavus in improving the efficacy of non-responsive patients is noteworthy. A larger-scale study is needed to determine the above findings.