Hyperlipidemia is closely related to oxidative stress caused by an imbalanced diet, and safe and effective dietary interventions have become a research hotspot in lipid management. To explore the feasibility and underlying mechanisms of flavonoids achieving synergistic lipid-lowering effects through probiotic-mediated in situ biotransformation, this study focused on a Lactobacillus pentosus (L. pentosus) 68-1 strain with disaccharidase activity. Combined fermentation with rutin was conducted, and the effects of different carbon sources on the strain''s glycosidase activity were evaluated. High-performance liquid chromatography (HPLC) was used to compare the quercetin content in the transformed products to obtain the optimal biotransformation process. The resulting transformed products were then fed to hyperlipidemic mice to systematically evaluate their effects on blood lipid levels and target organ morphology. The results showed that after 28 hours of fermentation, the conversion rate of rutin reached 29.81% and significantly enhanced antioxidant capacity. Compared to glucose as a carbon source, the activities of β-glucosidase and α-L-rhamnosidase increased by 84.39% and 27.65%, respectively, when the carbon source was optimized. In vivo experiments demonstrated that the combination of Lactobacillus pentosus 68-1 and rutin significantly improved mice’s blood lipid levels and target organ pathology. This study verified the superiority of the strategy “probiotic fermentation of flavonoids to produce highly active metabolites in situ,” providing a theoretical basis and experimental support for combined probiotic and dietary polyphenol lipid interventions.