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 in female mice results in significantly altered body composition that is associated with increases in energy expenditure, body temperature and physical activity. Male NPFFR2-/- mice show reduced energy intake and moderate reduction in adiposity, but no significant changes in energy expenditure or physical activity. Interestingly, when fed on a high-fat diet (HFD), both male and female NPFFR2-/- mice have greater weight gain that is contributed by increases in both lean and fat masses in males but primarily fat mass in females. Importantly, whereas male NPFFR2-/- consistently showed a reduced energy intake on HFD, both male and female NPFFR2-/- on HFD show significantly lower energy expenditure than that of WT, in contrast to observations under chow-fed condition. 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 both genders. 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.