Our previous studies showed that berberine (BBR) increases liver low-density lipoprotein (LDL) receptor expression in an extracellular signal-regulated kinase (ERK)-dependent manner. This study was designed to explore the upstream cellular signaling molecules recruited by BBR to activate the ERK mitogen-activated protein kinase (MAPK) cascade. Chemical inhibitors such as GW5074, manumycin A, and compound C or specific small interfering RNAs (siRNAs) were used in the blocking experiments; Western blot was used to determine the phosphorylation of kinases; real-time reverse transcriptase polymerase chain reaction (RT-PCR) was used to determine the expression level of LDL receptor mRNA. Our results indicate that BBR increases p-Raf-1 (ser338) level time and dose dependently in HL-7702 cells, but has no influence on Ras activity; the stimulating activities of BBR on Raf-1 signaling and LDL receptor expression can be blocked by GW5074 completely, but not by manumycin A, a Ras inhibitor. BBR activates hepatic Raf-1 signaling and up-regulates LDL receptor expression in a rat model of hyperlipidemia with no impact on liver Ras activity. Importantly, our results show that the stimulating activities of BBR on hepatic Raf-1 signaling and LDL receptor expression are totally blocked by compound C, a selective inhibitor of AMP-activated protein kinase (AMPK), and also by silencing its expression with siRNA. Taken together, our results demonstrate for the first time that BBR up-regulates LDL receptor expression through Ras-independent, but AMPK-dependent Raf-1 activation in liver cells. Our study will help to elucidate the molecular pharmacology of BBR and provide new scientific evidence for its clinical application.