Abstract: This study aimed to investigate the deastringency process of Rosa roxburghii juice fermented with a composite starter culture of Acetobacter and Rhizopus arrhizus, as well as the associated changes in nutritional components and metabolite profiles. Using raw R. roxburghii juice as the raw material and tannin degradation rate as the response value, the fermentation process was optimized through single-factor experiments and response surface methodology (RSM). Differences in nutritional composition and antioxidant activity before and after fermentation were analyzed, and untargeted LC-MS-based metabolomics was employed to examine metabolite variations. The results showed that the optimal fermentation conditions were as follows: glucose addition of 2.00%, initial pH of 3.5 (raw juice), inoculation with 0.325% R. arrhizus and 5.00% Acetobacter, and mixed fermentation for 3 days. Under these conditions, the tannin degradation rate reached 65.54%±2.31%. CCompared with raw juice, the fermented juice exhibited significantly decreased contents of reducing sugars, tannins, total phenolics, and L-ascorbic acid (P < 0.05), while the contents of polysaccharides and flavonoids increased significantly (P < 0.05); the antioxidant activity remained stable. A total of 1,926 metabolites were identified, among which 664 were screened as key differential metabolites, including 148 upregulated and 516 downregulated metabolites. The upregulated shikimic acid and phenylpropanoid compounds were mainly flavonoids. KEGG pathway enrichment analysis revealed that toluene degradation, D-amino acid metabolism, and amino acid biosynthesis pathways were significantly upregulated after fermentation. In the flavonoid biosynthesis pathway, typical astringent compounds such as pinocembrin, cyanidin, and myricetin were annotated, and showed significantly downregulated trends, confirming that composite probiotic fermentation can effectively reduce the astringency and improve the taste of R. roxburghii juice. Meanwhile, the increase in flavonoids contributed to the maintenance of stable antioxidant activity. This study provides a scientific basis for the development of functional beverages from Rosa roxburghii.