High-fat diets (HFD) disrupt lipid homeostasis, posing major public health risks. This study investigated the effect of ACA-DK, an insoluble dietary fiber derived from Antrodia camphorata, on HFD-induced dyslipidemia. We demonstrate that ACA-DK effectively alleviates HFD-induced dyslipidemia in mice, counteracting metabolic disorders, aberrant blood lipids, and weight gain. Mechanistically, ACA-DK modulates triglycerides, phosphatidylcholine, and phosphatidylethanolamine via glycerophospholipid/choline/linoleic acid metabolism pathways, while rectifying gut dysbiosis through selective reduction of pro-inflammatory genera (Oscillibacter, Ruminiclostridium, Negativibacillus, Ruminococcaceae and Helicobacter). Integrated analysis identifies Ruminococcaceae and Helicobacter as key mediators of ACA-DK’s lipid-regulatory effects, establishing microbiota-directed therapy as a strategy against dyslipidemia. We thus propose ACA-DK as a microbiota-directed dual-target therapy, simultaneously reprogramming host lipid metabolism and gut ecology to combat diet-induced metabolic diseases. These findings suggest that ACA-DK is a promising prebiotic dietary fiber for ameliorating HFD-induced lipid metabolic disorders, with potential for future development into functional foods or supplements.