Non-alcoholic fatty liver disease (NAFLD) is a major public health concern worldwide. The aim of the present study was to observe the effect of diosgenin on NAFLD and investigate the underlying mechanisms. Diosgenin treatment increased the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) in HepG2 cells. Diosgenin significantly inhibited high glucose (HG)-induced triglyceride (TG) accumulation and sterol regulatory element‑binding protein-1c (SREBP-1c) mRNA increase in HepG2 cells, which were partially abolished by the AMPK inhibitor compound C. Diosgenin also significantly inhibited the increase of liver X receptor (LXR) α mRNA induced by HG or T0901317. However, T0901317‑induced upregulation of LXRα and SREBP-1c mRNA was not blocked by compound C. Following a high-fat diet for 16 weeks, the body and liver weights of the experimental rats were significantly increased, but this effect was significantly suppressed by diosgenin. Diosgenin and fenofibrate ameliorated lipid deposition in the liver and reduced the increase of hepatic TG content. Diosgenin significantly decreased the alanine aminotransferase (ALT) level, whereas fenofibrate significantly increased the ALT and aspartate aminotransferase levels. Diosgenin also increased AMPK and ACC phosphorylation and suppressed LXRα in the liver. In conclusion, the results of the present study suggested that diosgenin is a potential agent for preventing the development of NAFLD through the AMPK and LXR signaling pathways.