Non-alcoholic fatty liver disease (NAFLD) has becomeserious liverdisease all over the world. At present, NAFLD caused by high calorieand fat diet is increasing. Calsyntenin-3 (Clstn3) is a transmembraneprotein that has recently been found to participate in lipid energymetabolism. But whether Clstn3 affects NAFLD lipid metabolism hasnot been analyzed. We stimulate the mice primary hepatocytes (MPHs)with oleic acid and palmitic acid (OA & PA) to establish a cellmodel. Then, potential targets, including Clstn3 gene, were validatedfor improving lipid metabolism disorder in NAFLD model mice (HFD anddb/db) by silencing and overexpressing hepatic Clstn3. Moreover, theeffects of Clstn3 on lipid homeostasis were determined by functionaldetermination, triglyceride (TG) levels, total cholesterol (TC) levels,ELISA, and qRT-PCR detection. Our results displayed that Clstn3 wasdecreased in the NAFLD mice model. Also, overexpression of Clstn3improved lipid metabolism disorders, gluconeogenesis, and energy homeostasisand reduced liver injury, inflammation, and oxidative stress injury.However, opposite results were obtained in Clstn3-silencing mice,suggesting that the Clstn3 gene is closely related to lipid metabolismdisorder in NAFLD. RNAseq expression demonstrated that Farnesoid XReceptor (FXR) expression was increased after overexpression of Clstn3.Clstn3 supplementation in FXRKO mice can improve the dysfunction causedby insufficient FXR, suggesting that Clstn3 can improve the NAFLDlipid metabolism disorder to some extent through FXR, which may providea new method for the treatment of NAFLD.