Description: Hyperhomocysteinemia (HHcy) is associated with liver diseases such as fatty liver and hepatic fibrosis; however, the underlying mechanism is still largely unknown. The current study aimed to explore the signaling pathway involved in HHcy-induced hepatic steatosis. C57BL/6 mice were fed a high-methionine diet (HMD) for 4 and 8 weeks to establish the HHcy mouse model. Compared to a chow diet, the HMD induced hepatic steatosis and elevated hepatic expression of CD36, a fatty acid transport protein. The increased CD36 expression was associated with activation of the aryl hydrocarbon receptor (AHR). In primary cultured hepatocytes, high levels of homocysteine (Hcy) treatment upregulated CD36 and increased subsequent lipid uptake; both were significantly attenuated by siRNA knockdown of CD36 and AHR. Chromatin immunoprecipitation assay revealed that Hcy promoted the binding of AHR to the CD36 promoter, and transient transfection assay demonstrated markedly increased activity of the AHR response element by Hcy, which was ligand-dependent. Mass spectrometry revealed significantly increased hepatic content of lipoxin A 4 (LXA 4 ), a metabolite of arachidonic acid, in HMD-fed mice. Further, over-expression of 15-oxoprostaglandin 13-reductase 1, a LXA 4 inactivation enzyme, inhibited Hcy-induced AHR activation, lipid uptake and lipid accumulation. Moreover, LXA 4 -induced upregulation of CD36 and lipid uptake was inhibited by AHR siRNA in vitro in hepatocytes. Finally, treatment with an AHR antagonist reversed the HHcy-induced lipid accumulation by inhibiting the AHR-CD36 pathway in mice. In conclusion, our results strongly suggest that HHcy activated the AHR-CD36 pathway by increasing hepatic LXA 4 content, which resulted in hepatic steatosis. This article is protected by copyright. All rights reserved.