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The gut microbiota influences host metabolism and health, impacting diseases. Research into how diet affects gut microbiome dynamics in model organisms is crucial but underexplored. Herein, we examined how dietary adenine affects uric acid levels and the gut microbiota over five generations of Drosophila melanogaster . Wild-type W1118 flies consumed diets with various adenine concentrations (GC: 0%, GL: 0.05%, and GH: 0.10%), and their gut microbiota were assessed via Illumina MiSeq sequencing. Adenine intake significantly increased uric acid levels in the GH group > the GC group. Despite no significant differences in the alpha diversity indices, there were significant disparities in the gut microbiota health index (GMHI) and dysbiosis index (MDI) among the groups. Adenine concentrations significantly altered the diversity and composition of the gut microbiota. High adenine intake correlated with increased uric acid levels and microbial population shifts, notably affecting the abundances of Proteobacteria and Firmicutes. The gut microbiota phenotypes included mobile elements, gram-positive bacteria, biofilm-forming bacteria, and gram-negative bacteria. The significantly enriched KEGG pathways included ageing, carbohydrate metabolism, and the immune system. In conclusion, adenine intake increases uric acid levels, alters gut microbiota, and affects KEGG pathways in Drosophila across generations. This study highlights the impact of dietary adenine on uric acid levels and the gut microbiota, providing insights into intergenerational nutritional effects., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Yin, Tong, Wang, Wei, Qin, Wu, Zhang, Guan and Qiu.)