Human serum paraoxonase 1 (PON1), a calcium-dependent ester hydrolase, protects against the oxidative modification of low-density lipoprotein (LDL) and is a major anti-atherosclerotic component of high-density lipoprotein (HDL). Graptopetalum paraguayense, a folk herbal medicine commonly used in Taiwan, has antioxidative, anti-inflammatory, anti-hypertensive, and anti-atherogenic properties. The effects of G. paraguayense on the activity and/or expression of PON1 were examined using various extracts of the plant; extracts were made in water (GPWE), 50% ethanol (GP50E), and 95% ethanol (GP95E). Of these extracts, GP50E was found to be the most effective at increasing the function and expression of PON1 in a human hepatoma HepG2 cell line. Data from electrophoretic mobility shift assays and promoter-reporter luciferase analyses demonstrated that the DNA binding activity and transactivation ability of NF-κB were enhanced by GP50E. Treatment with NF-κB inhibitors, pyrrolidine dithiocarbamate, and BAY 11-7082 significantly attenuated GP50E-induced PON1 production and NF-κB transactivation activity. In addition, GP50E increased the levels of phosphorylated protein kinase B (PKB/AKT). Pharmacological inhibition of AKT by LY294002 effectively suppressed NF-κB activation and PON1 gene expression, suggesting that AKT was an upstream regulator of GP50E-mediated biological events. Overall, the results show that GP50E up-regulated PON1 gene expression via an AKT/NF-κB-dependent signaling pathway in human hepatoma HepG2 cells. This observation led to the conclusion that the anti-atherogenic characteristics of G. paraguayense are modulated, at least in part, via the up-regulation of hepatocyte PON1 gene expression.