Non-alcoholic fatty liver disease (NAFLD) is a most common liver disorder characterized by accumulation of fat in the liver and currently there is no approved treatment for it. Obesity and diabetes being leading cause of NAFLD, compounds having anti-obesity activity and potential to reduce insulin resistance are considered suitable candidate for NAFLD treatment. In this study, we checked effect of vitexin, a naturally occurring flavonoid, on high fat diet (HFD) induced NAFLD in C57BL/6J mice. In presence of vitexin, significant reduction in body and liver weight, triglyceride and cholesterol content in serum and liver was observed. Serum Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) levels were reduced significantly by vitexin which were elevated in HFD group whereas serum lipase activity remained unchanged. Vitexin suppressed de novo lipogenesis by downregulating expression of Peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein-α (C/EBP-α), sterol regulatory element-binding protein-1c (SREBP-1c), Fatty acid synthase (FAS) and Acetyl-CoA Carboxylase (ACC). Additionally, it also enhanced fatty acid oxidation and lipolysis by upregulating Peroxisome proliferator-activated receptor α (PPAR-α), carnitine palmitoyltransferase-1a (CPT-1a) and Adipose triglyceride lipase (ATGL). Inhibition of lipogenesis and activation of lipolysis and fatty acid oxidation by vitexin was found to be mediated by activation of AMP-activated protein kinase (AMPK). Vitexin also improved insulin signalling by activating insulin receptor substrate-1 (IRS-1) and its downstream target AKT. AMPK activation of vitexin was possibly through binding of vitexin to leptin receptor (LepR) which was confirmed by molecular docking studies and by observed enhanced expression of LepR. Thus, we propose that vitexin alleviates NAFLD by activating AMPK possibly by binding to LepR.