The neuropeptide FF receptor 2 (NPFFR2) is highly expressed in the hypothalamus where it is activated by a set of RFamide peptides, however, its physiological function is unclear. Here we show that lack of NPFFR2 results in significantly altered body composition that is associated with increases in energy expenditure, body temperature and physical activity. On the other hand, food intake was not significantly altered in NPFFR2-/- versus WT mice. Interestingly, when fed on a high-fat diet (HFD), NPFFR2-/- mice showed greater weight gain and fat gain, and significantly reduced energy expenditure compared to WT mice. When energy expenditure from HFD and chow studies are compared, there are significant interactions between genotype and diet effects, i.e. HFD increases energy expenditure in both NPFFR2-/- and WT mice, however this increase is significantly less in NPFFR2-/- than that in WT mice, suggesting an impaired diet-induced adaptive thermogenesis by lack of NPFFR2 signalling. In support, HFD significantly increased UCP-1 and PGC-1α levels in the brown adipose tissue of WT mice but not in that of NPFFR2-/- mice. The mechanism behind NPFFR2 control of energy expenditure and adaptive thermogenesis is likely to involve hypothalamic neuropeptide Y pathways, since the HFD-induced decrease in hypothalamic NPY expression observed in WT is absent in NPFFR2-/- mice. Taken together, these data demonstrate that NPFFR2 signalling plays important roles in the regulation of energy homeostasis and diet-adaptive thermogenesis, which may involve hypothalamic NPY pathways.