Background: Our team has previously demonstrated in two prospective studies that the live bacterial product CBM588 may enhance clinical outcomes in patients with mRCC (Ebrahimi et al ASCO 2023, Dizman et al Nature Med 2022). In the current study, we sought to determine if gut microbial functionality is associated with clinical outcomes in patients with mRCC treated with cabo/nivo with or without CBM588. Methods: Pts ≥18 yrs old with histologically verified (clear-cell, papillary, or sarcomatoid component) mRCC and no prior systemic therapy for metastatic disease were enrolled and randomized 1:2 to receive either cabo/nivo at the standard dose/schedule alone or with CBM588 dosed at 80mg PO BID. Whole metagenome sequencing was performed on stool specimens collected at baseline and week 12 of treatment. Taxonomic profiling was conducted using MetaPhlAn 4, and functional profiling was performed using HUMAnN 3. HUMAnN 3 annotates open reading frames and provides highly accurate information on metabolic pathways and other molecular functions from metagenomic or metatranscriptomic sequencing data. The ANCOM-BC was used to detect the taxonomic/genetic features with differential abundance between two time-points within the same treatment arm. Results: A total of 30 (20:10 M:F) pts were enrolled with a median age of 65 (36-84). 5 pts (17%) had sarcomatoid features, and 2 pts (7%) had predominant papillary histology. Objective response was achieved in 20% and 65% of the pts in the cabo/nivo and cabo/nivo/CBM588 arm, respectively. Significant changes in 9 metabolic pathways (1 upregulation, 8 downregulation) in the control arm and 7 metabolic pathways (2 upregulation, 5 with downregulation) in the experimental arm were identified. Superpathways of biosynthesis of different forms of menaquinole, a reversible redox component of the electron transfer chain, were depleted with cabo/nivo treatment. In contrast, the biosynthesis of menaquinol-8 and 1,4 dihydroxy-6-naphthoate (an intermediate of the menaquinone pathway) were upregulated in cabo/nivo/CBM588 arm. Conclusions: Our interrogation of metabolic dynamics and pathways in patients receiving CBM588 suggests key differences in biosynthesis pathways of menaquinone between control and experimental arms. Menaquinones (vitamin K2 derivatives) have been previously reported to induce apoptosis in many cancer cell types and also increase the objective response rate to sorafenib in patients with hepatocellular carcinoma. Our findings provide mechanistic evidence for the effect of the addition of CBM588 to cabo/nivo on gut microbiome function and the resultant improvement in clinical outcomes in mRCC, potentially through enhancing the enteric production of vitamin K2. Clinical trial information: NCT05122546.