Background: The gut microbiome is emerging as a key regulator of the immune system during immunotherapy. However, the effect of modulating the gut microbiome in patients (pts) with cancer refractory to immunotherapy remains largely unknown. We aimed to evaluate whether the fecal microbiota transplantation (FMT) could help overcome resistance in pts with advanced solid cancer refractory to anti-PD-(L)1 inhibitors and identify specific commensal bacteria that contribute to the efficacy of FMT (NCT04264975). Methods: This is a prospective, single-arm clinical trial of FMT plus anti-PD-(L)1 inhibitor in pts with advanced solid cancer refractory to anti-PD-(L)1 inhibitors. The key eligibility criteria for donors included ongoing durable complete or partial response ≥ 6 months with anti-PD-(L)1 monotherapy for advanced solid tumors. FMT was performed using colonoscopy, followed by continuation or reintroduction of anti-PD-(L)1 inhibitor until unacceptable toxicity or disease progression. Repeated FMT from the same or different donors was allowed. Results: From Jan. 2019 to Aug. 2020, 13 pts with metastatic gastric cancer (GC) (n=4), esophageal squamous cell carcinoma (ESCC) (n=5), and hepatocellular carcinoma (HCC) (n=4) were enrolled; male (77%), median age=60 yrs (range, 38-76), and median line of prior systemic therapy=3 (range, 2-5). All had confirmed disease progression on nivolumab monotherapy with primary (46.2%) or secondary resistance (53.8%), and underwent FMT with continued nivolumab. There were six FMT donors (HCC [n=4], GC [n=1], ESCC) [n=1], who had maintained (CR; n=4) or (PR; n=2) with nivolumab or pembrolizumab monotherapy. Of the 13 recipients, five showed SD and one achieved PR after FMT with a disease control rate of 46.2% (6/13) and an objective response rate of 7.7% (1/13). Recipient #7 (R7), who had metastatic HCC with primary resistance to nivolumab, initially showed PD to the 1st FMT from donor #1, but achieved PR after the 2nd FMT from donor #5. Clinical response was accompanied by an increase in levels of cytotoxic T cells in the blood and tumor microenvironment, immune cytokines, and the relative abundance of a new species derived from donor #5 showing 97% whole genome nucleotide sequence similarity with Prevotella sp. Marseille-P4119. We isolated this species from feces of R7 and preclinical experiments showed that treatment with this species activated human CD4+ and CD8+ T cells with increased IFN-γ secretion, and suppressed tumor growth in a syngeneic mouse model by enhancing tumor infiltration of cytotoxic T cells. Moreover, the combination treatment with anti-PD-1 and this species reduced the tumor volume more than with anti-PD-1 alone. Conclusions: FMT containing the effective microbiota could overcome resistance to anti-PD-1 inhibitor by modulating the tumor microenvironment in advanced solid cancer pts. Clinical trial information: NCT04264975.