The gut microbiomes communicate with the kidney and may play a crucial role in the development of anemia in patients with chronic renal failure (CRF). However, the alterations in microbiota and their association with functional metabolites remain unclear. We performed metagenomics and untargeted metabolomics in a cohort of 30 patients with anemia of CRF and 20 healthy controls (HCs) to identify the characteristics of the gut microbiome and explore its potential interactions with the host. Decreased microbiota diversity and significant compositional differences were observed in the patients with anemia of CRF. We identified six gut microbiotas significantly changed in the patients with anemia of CRF, particularly , and , which were closely correlated with hemoglobin (Hb) levels and estimated glomerular filtration rate (eGFR). These changes were accompanied by functional alterations in distinctive microbial pathways. Further fecal and serum metabolomics revealed fecal 12-KETE-LTB4 in arachidonic acid metabolism, uracil and L-aspartic acid in beta-alanine metabolism, gulonic acid in ascorbate and aldarate metabolism, accompanied by the top 15 differential serum metabolites that were closely correlated with Hb levels. Furthermore, we observed a complex co-occurrence between anemia of CRF-related gut microbiota species and the characterized metabolites. Moreover, a non-invasive model incorporating and , combined with fecal 12-KETE-LTB4, uracil, L-aspartic acid, and gulonic acid, distinguished the patients with anemia of CRF from HCs (area under the curve: 0.879). Collectively, our results suggest that a disordered gut microbiome associated with functional metabolites may be a non-invasive diagnostic and therapeutic target for anemia of CRF.Anemia is a prevalent complication in patients with chronic renal failure (CRF), which is associated with a high burden of morbidity and adverse clinical outcomes. Various evidence suggests that gut microbiota dysbiosis may contribute to the pathogenesis of anemia in CRF, although the mechanism is still obscure. This work provides substantial evidence identifying the specific characteristics of the gut microbiomes accompanied by functional alterations in anemia of CRF. We highlight the intricate interactions among the anemia of CRF-related gut microbiome and the functional metabolites, which may regulate toxic accumulation, oxidative stress, and immune-inflammatory responses to induce and exacerbate anemia in patients with CRF. Furthermore, we found that evaluating the gut microbiota and fecal metabolites in combination might be a non-invasive prognostic indicator of CRF-induced anemia. These findings provide important insights into the role of gut microbiota in the mechanism of anemia in CRF.CLINICAL TRIALSThis study is registered with ClinicalTrials.gov as NCT05543291.