The non-alcoholic fatty liver disease (NAFLD) has become a serious medical problem and an increasing threat to public health. It is characterized by the abnormal fat accumulation in liver without excessive alcohol intake. The concurrent NAFLD might up-regulate the risk of chronic kidney disease as well as the mortality rate. Though various drugs have been investigated to attenuate NAFLD, further study is still necessary to find new therapeutic strategy and to reveal the underlying molecular mechanism. In the present study, NAFLD animal models were induced by feeding with high fat (HF) diet for 8 weeks. Alpinetin (ALP) was given to mice for another 8 weeks together with HF. Hepatic and renal function, oxidative stress, inflammatory response and lipid metabolism were calculated. And human liver cells of HL-7702 were cultured with high fructose (5mM) with or without ALP. The findings indicated that ALP down-regulated lipid accumulation in liver tissue samples. The higher inflammatory score induced by HF in liver and renal were reduced by ALP. HF-triggered oxidative stress was inhibited in ALP-treated groups, as evidenced by enhanced SOD1/HO-1/Nrf-2 expressions and reduced thioredoxin-interacting protein (TXNIP)/xanthine oxidase (XO) levels. ALP also suppressed inflammatory response by decreasing pro-inflammatory cytokines through inactivating toll-like receptor 4-nuclear factor kappa B (TLR4-NF-κB) pathway. The anti-oxidant and anti-inflammatory effects of ALP were confirmed in HL-7702 cells. Further, abnormal lipid metabolism caused by HF was alleviated by ALP, which was associated with the decreased Stearoyl-CoA desaturase 1 (SCD1), fatty acid synthase (FAS), sterol element regulatory binding protein 1c (SREBP-1c), Liver X Receptor (LXR)-α, elongases of very long-chain fatty acids (Elovl)-2, p-insulin receptor substrate 1 (IRS1) expressions, and increased PPARα levels. Taken together, the results above indicated that ALP could suppress oxidative stress, reduce inflammatory response and attenuate lipid metabolism, preventing NAFLD.