Obesity is a growing epidemic with limited effective treatments. The neurotrophic factor glial cell line derived neurotrophic factor (GDNF) was recently shown to enhance β-cell mass and improve glucose control in rodents. It's role in obesity is, however, not well characterized. In this study, we investigated the ability of GDNF to protect against high fat diet (HFD)-induced obesity. GDNF transgenic (Tg) mice, that over express GDNF under the control of the glial fibrillary acidic protein promoter, and wild-type (WT) littermates were maintained on a HFD or regular rodent diet (RD) for 11 weeks, and weight gain, energy expenditure and insulin sensitivity monitored. Differentiated mouse brown adipocytes and 3T3-L1 white adipocytes were used to study the effects of GDNF in vitro. Tg mice resisted the HFD-induced weight gain, insulin resistance, dyslipidemia, hyperleptinemia and hepatic steatosis seen in WT mice despite similar food intake and activity levels. They exhibited significantly (P<0.001) higher energy expenditure than WT mice and increased expression in skeletal muscle and brown adipose tissue of peroxisome proliferator activated receptor-α and β1- and β3-adrenergic receptor genes which are associated with increased lipolysis and enhanced lipid β-oxidation. In vitro, GDNF enhanced β-adrenergic-mediated cAMP release in brown adipocytes and suppressed lipid accumulation in differentiated 3T3L-1 cells through a p38MAPK signaling pathway. Our studies demonstrate a novel role for GDNF in the regulation of high fat diet-induced obesity through increased energy expenditure. They show that GDNF and its receptor agonists may be potential targets for the treatment or prevention of obesity.