SummaryThe decision to sleep requires integration of information about the internal state of the animal and its immediate external environment. Sleep circuitry therefore evolved to have both dedicated and context-dependent modulatory neuronal elements [1-3]. Identifying these important subcircuits and understanding the molecular mechanisms regulating their roles is a major challenge for the sleep field. microRNAs, non-coding RNA transcripts which posttranscriptionally control gene expression [4], have been implicated in sleep regulation [5-10]. Our previous screen [11] identified 25 sleep-regulating microRNAs in Drosophila melanogaster , including the developmentally-important [12-15] microRNA bantam ( ban ). Here we show that ban acts in the adult brain to promote early nighttime sleep through a population of glutamatergic neurons that is intimately involved in applying contextual information to behaviors [16]- the γ5β′2a/β′2mp/β′2mp_bilateral Mushroom Body Output Neurons (MBONs). GCaMP calcium imaging revealed that bantam inhibits the neural activity of these cells during the early night, but not the day. Blocking synaptic transmission in these MBONs rescued the effect of ban knockdown. Together these results suggest ban promotes early night sleep via inhibition of the γ5β′2a/β′2mp/β′2mp_bilateral MBONs. RNAseq identifies Kelch and CCHamide-2 receptor as mediators of this role. These experiments establish a new role for bantam as an active regulator of sleep and neural activity in the adult fly brain. Notably, the gene for BRS3 (Bombesin receptor subtype-3), the human ortholog of CCHa2-R, hosts an MRE for the mammalian ortholog of ban , miR-450b-3p [17-19]. The regulation of orexin neurons by this receptor [20] implies this pathway is highly conserved.