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Purpose: Multiple factors, such as dietary patterns, pharmaceutical interventions, and exposure to harmful substances, possess the capacity to influence gut microbiota composition. Gut microbiota dysbiosis has emerged as a significant contributor to the progression of chronic kidney disease (CKD) and its associated complications. By comprehending the intricacies of the intestinal microbiota, this research endeavor holds the potential to offer novel perspectives on potential strategies for mitigating CKD progression. Methods: In this retrospective analysis, we assessed gut microbiota composition in CKD patients. Fecal samples were collected from a cohort of 44 patients with stage 3–4 CKD, alongside a control group consisting of 132 healthy volunteers. Subsequently, 16 s rDNA sequencing was conducted to examine the composition of the gut microbiota. Results: Our findings revealed significant alterations in the diversity of intestinal microbiota in fecal samples between patients with stage 3–4 CKD and healthy subjects. Among the 475 bacterial genera, 164 were shared, while 242 dominant genera were exclusive to healthy subjects and 69 to CKD stages 3–4 samples. Notably, healthy volunteers exhibited a prevalence of intestinal Firmicutes and Bacteroidetes, whereas stage 3–4 CKD patients displayed higher abundance of Proteobacteria and Actinobacteria. The presence of uncultured Coprobacillus sp. notably contributed to distinguishing between the two groups. ROC curve analysis identified distinct microbiota with superior diagnostic efficacy for discriminating stage 3–4 CKD patients from healthy individuals. Metabolic pathway analysis revealed differing dominant pathways between the two groups—the NADH dehydrogenase pathway in healthy individuals and the phosphate acetyltransferase pathway in stage 3–4 CKD patients. Moreover, the CKD cohort displayed a higher proportion of Gram-negative bacteria and facultative anaerobes. Conclusions: In conclusion, our study underscores the profound influence of gut microbiota dysbiosis on CKD progression. The distinct microbial profiles observed in CKD patients highlight the potential efficacy of microbiota-based interventions in mitigating CKD advancement. [ABSTRACT FROM AUTHOR]