Abstract: The processing of highland barley generates a significant quantity of bran, which is abundant in soluble dietary fiber (SDF), yet this by-product remains largely underutilized. Existing research indicates that SDF has the potential to ameliorate lipid metabolic disorders through the regulation of bile acid metabolism. However, the potential of highland barley bran-derived SDF to modulate bile acid metabolism and the underlying mechanisms remain unexplored. This study utilized 5-week-old SPF-grade male C57BL/6J mice fed a high-fat diet to investigate the effects of highland barley bran SDF intervention on total bile acid (TBA) levels in serum and liver tissues. The study also examined the hepatic bile acid metabolic profile and identified differential bile acids responsive to highland barley bran SDF treatment. Additionally, RT-qPCR and Western blot analyses were conducted to evaluate the expression levels of bile acid metabolism-related genes and proteins in both the liver and ileum, aiming to preliminarily elucidate the underlying mechanisms by which highland barley bran SDF regulated bile acid metabolism. The results demonstrated that the TBA levels in both serum and hepatic tissues of high-fat diet-fed mice were elevated following intervention with highland barley bran SDF. Through targeted metabolomics analysis, five potential biomarkers responsive to highland barley bran SDF treatment were identified. Highland barley bran SDF regulated bile acid metabolism in mice by inhibiting the activity of the ileal Farnesoid X Receptor (FXR) to promote hepatic primary bile acid biosynthesis, while simultaneously activating the ileal G Protein-Coupled Bile Acid Receptor 5 (TGR5) to accelerate bile acid transport. This study provides a theoretical basis for understanding the lipid-lowering effects of SDF derived from highland barley bran and offers valuable insights for the high-value utilization of highland barley bran as well as the development of lipid-lowering functional foods.