Angiopoietin-like protein 4 (ANGPTL4) is a circulating protein predominantly produced from fat tissue and liver. Recent data from others and our laboratory (PNAS, 2005, 102:6086-91) have demonstrated this protein to be an important player in energy metabolism and insulin sensitivity. However, the molecular mechanisms underlying its metabolic actions remain elusive. In this study, we have employed two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) technique to study the protein profiles in the livers of db/db mice treated with or without ANGPTL4. 118 proteins were found to be up- or down-regulated in db/db mice comparing with those of lean mice. Adenovirus-mediated over-expression of ANGPTL4 could reverse a large portion of the up- or down-regulated proteins to control levels. Especially, a number of mitochondria proteins were down-regulated by ANGPTL4 to a great extent. Chronic treatment with ANGPTL4 resulted in an elevated activity of mitochondria respiratory chain complexes II-III and IV in db/db mice. Additionally, several key enzymes in the methionine/homocysteine metabolic cycle were found to be increased in db/db diabetic mice but decreased by ANGPTL4 treatment. Consistently, HPLC analysis revealed that ANGPTL4 could significantly restore the augmented S-adenosylmethionine levels and S-adenosylmethionine/S-adenosylhomocysteine ratios in livers of db/db mice. In summary, our results suggest that ANGPTL4 might elicit its metabolic effects through modulating the mitochondria functions and methionine metabolic cycles in the liver tissue.