The short neuropeptide F (sNPF), a neuropeptide in the central nervous system (CNS) of Drosophila melanogaster, is expressed in a large population of diverse neurons of brain. Most of these neurons are intrinsic interneurons of the mushroom bodies, which are the most prominent insect bilateral CNS structures that regulate memory and sleep. However, its role in sleep regulation still remains elusive. Here, we showed that sNPF-deficient female and male flies exhibit sleep enhancement with an increase of sleep bout duration. Loss of function of sNPF and sNPFR1 also elevated sleep. Moreover, the homeostatic regulation of flies after sleep deprivation was disrupted by aberrant sNPF signaling, since sleep deprivation increased transcription of sNPF and wakefulness at night in control flies but not in the sNPF mutant flies, suggesting that sNPF autoregulation plays an important role in sleep homeostasis. We further verified that sNPF signal elevated cAMP levels, and subsequently activated the downstream CREB transcription factor. The duration of sleep was found to be inversely related to cAMP signaling and CREB activity in the mushroom bodies. Thus, we concluded that sleep might be regulated by sNPF through modulating the cAMP-PKA-CREB signal pathway in vivo.