Rhizoma Coptidis (RC), the dried rhizome of Coptis Chinensis Franch., can dispel dampness and heat within the body and has been traditionally used for the treatment of cardiovascular disease (CVD)-associated problems including hyperlipidemia in China. Berberine (BBR) is the main active component of RC, which has been shown to possess significant therapeutic potential. However, only 0.14% of BBR is metabolized in the liver, and the extremely low bioavailability (<1%) and blood concentration of BBR in experimental and clinical settings is insufficient to achieve the effects as observed under in vitro conditions, which imposes challenges to explain its excellent pharmacological actions. Intense efforts are currently being devoted to defining its specific pharmacological molecular targets, while the exploration from the perspective of its pharmacokinetic disposition has rarely been reported to date, which could hardly make a comprehensive understanding of its hypolipidemic enigma.This study made a pioneering endeavor to unveil the hypolipidemic mechanism of BBR from RC focusing on its unique intestines-erythrocytes-mediated bio-disposition.The fate of BBR in intestines and erythrocytes was probed by a rapid and sensitive LC/MS-IT-TOF method. To analyze the disposition of BBR, a reliable HPLC method was subsequently developed and validated for simultaneous determination of BBR and its key active metabolite oxyberberine (OBB) in whole blood, tissues, and excreta. Meanwhile, the enterohepatic circulation (BDC) of BBR and OBB was verified by bile duct catheterization rats. Finally, lipid overloading models of L02 and HepG2 cells were employed to probe the lipid-lowering activity of BBR and OBB at in vivo concentration.The results showed that BBR underwent biotransformation in both intestines and erythrocytes, and converted into the major metabolite oxyberberine (OBB). The AUC0-t ratio of total BBR to OBB was approximately 2:1 after oral administration. Besides, the AUC0-t ratio of bound BBR to its unbound counterpart was 4.6:1, and this ratio of OBB was 2.5:1, indicative of abundant binding-type form in the blood. Liver dominated over other organs in tissue distribution. BBR was excreted in bile, while the excretion of OBB in feces was significantly higher than that in bile. Furthermore, the bimodal phenomenon of both BBR and OBB disappeared in BDC rats and the AUC0-t was significantly lower than that in the sham-operated control rats. Interestingly, OBB significantly decreased triglycerides and cholesterol levels in lipid overloading models of L02 and HepG2 cells at in vivo-like concentration, which was superior to the prodrug BBR.Cumulatively, BBR underwent unique extrahepatic metabolism and disposition into OBB by virtue of intestines and erythrocytes. BBR and OBB were mainly presented and transported in the protein-bound form within the circulating erythrocytes, potentially resulting in hepatocyte targeting accompanied by obvious enterohepatic circulation. The unique extrahepatic disposition of BBR via intestines and erythrocytes conceivably contributed enormously to its hypolipidemic effect. OBB was the important material basis for the hypolipidemic effect of BBR and RC.Copyright © 2023 Elsevier B.V. All rights reserved.