To investigate the possible role of eukaryotic translation initiation factor 4E binding protein 2 (4E-BP2) in metabolism and energy homeostasis, high-fat diet induced obese mice were treated with a 4E-BP2 specific antisense oligonucleotide (ASO) or a control ASO at a dose of 25 mg/kg BW or with saline twice a week for 6 weeks. 4E-BP2 ASO treatment reduced 4E-BP2 levels by > 75% in liver, white (WAT) and brown fat (BAT) tissues. Treatment did not change food intake but lowered body weight by about 7% and body fat content by about 18%. Treatment decreased liver triglyceride content by > 50%, normalized plasma glucose and insulin levels and reduced glucose excursion during glucose tolerance test. ASO-treated mice showed > 8.5% increase in metabolic rate, > 40 % increase in UCP1 levels in BAT, > 45% increase in AR-₃ mRNA and 40-55% decrease in mDIC, FAS, and DGAT2 mRNA levels in WAT. ASO-transfected mouse hepatocytes showed an increased fatty acid oxidation rate and a decreased TG synthesis rate. In addition, ASO-treated mice demonstrated about 60% and 29% decrease in hepatic G6Pase and PEPCK mRNA, respectively, implying decreased hepatic glucose output. Furthermore, increased phosphorylation of Akt^Ser473 in both liver and fat of ASO-treated mice and increased GLUT4 levels in plasma membrane in WAT of the ASO-treated mice were observed, indicating enhanced insulin signaling and increased glucose uptake as a consequence of reduced 4E-BP2 expression. These data demonstrate for the first time that peripheral 4E-BP2 plays an important role in metabolism and energy homeostasis.