BACKGROUND It is well known that nonalcoholic fatty liver disease (NAFLD) is associated with insulin resistance (IR). LB100, a serine/threonine protein phosphatase 2A (PP2A) inhibitor, is closely related to IR. However, there is little data regarding its direct influence on NAFLD. OBJECTIVE To elucidate the effect and underlying mechanism of LB100 in NAFLD. METHODS After 10 wk of high fat diet (HFD) feeding, male C57BL/6 mice were injected intraperitoneally with vehicle or LB100 for an additional 6 wk (three times a week). The L02 cell line was treated with LB100 and free fatty acids (FFAs) for 24 h. Hematoxylin and eosin and oil red O staining were performed for histological examination. Western blot analysis was used to detect the protein expression of Sirtuin 1 (Sirt1), total and phosphorylated AMP-activated protein kinase α (AMPKα), and the proteins involved in lipogenesis and fatty acid oxidation. The mRNA levels were determined by qPCR. Pharmacological inhibition of AMPK was performed to further examine the exact mechanism of LB100 in NAFLD. RESULTS LB100 significantly ameliorated HFD-induced obesity, hepatic lipid accumulation and hepatic injury in mice. In addition, LB100 significantly downregulated the protein levels of acetyl-CoA carboxylase, sterol regulatory element-binding protein 1 and its lipogenesis target genes, including stearoyl-CoA desaturase-1 and fatty acid synthase, and upregulated the levels of proteins involved in fatty acid β-oxidation, such as peroxisome proliferator-activated receptor α (PPARα), peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), carnitine palmitoyltransferase 1α, acyl-CoA oxidase 1 and uncoupling protein 2, as well as the upstream mediators Sirt1 and AMPKα in the livers of HFD-fed mice. In vitro, LB100 alleviated FFA-induced lipid accumulation in L02 cells through the AMPK/Sirt1 signaling pathway. Further studies showed that the curative effect of LB100 on lipid accumulation was abolished by inhibiting AMPKα in L02 cells. CONCLUSIONS PP2A inhibition by LB100 significantly ameliorates hepatic steatosis by regulating hepatic lipogenesis and fatty acid oxidation via the AMPK/Sirt1 pathway. LB100 may be a potential therapeutic agent for NAFLD.