Circadian rhythms in behaviour and physiology exist widely in animals, plants, fungi and cyanobacteria. Although much work has been carried out to characterize the endogenous clock circuit, the output signals coupling the circadian pacemaker to behaviour and physiology remain elusive. Here, we show that neuropeptide F (NPF), a homologue of mammalian neuropeptide Y, and its G protein-coupled receptor NPFR1 regulate the locomotor rhythm in Drosophila melanogaster. Flies with loss of function in NPF or NPFR1 were unable to ramp up their activity before lights off under light : dark (LD) cycles, and oscillations in npf/NPF and npfr1/NPFR1 were found to correlate temporally with the locomotor rhythm. Furthermore, NPF is expressed in clock neurones including dorsolateral neurones (LNds) and ventrolateral neurones (LNvs), whereas NPFR1 is expressed in dorsal neurone 1 (DN1) and LNds. These results show that NPF signalling is involved in the circadian locomotor rhythm in LD cycles.