The hypothalamic neuropeptide, orexin (or hypocretin), is implicated in numerous physiology and behavioral functions, including affective states such as depression and anxiety. The underlying mechanisms and neural circuits through which orexin modulates affective responses remain unclear. The objective of the present study was to test the hypothesis that the serotonin (5-HT) system of the dorsal raphe nucleus (DRN) is a downstream target through which orexin potentially manifests its role in affective states. Using a diurnal rodent, the Nile grass rat (Arvicanthis niloticus), we first characterized the expression of the orexin receptors OX1R and OX2R in the DRN using in situ hybridization. The results revealed distinct distributions of OX1R and OX2R mRNAs, with OX1R predominantly expressed in the dorsal and lateral wings of the DRN that are involved in affective processes, while OX2R was mostly found in the ventral DRN that is more involved in sensory-motor function. We next examined how the orexin-OX1R pathway regulates 5-HT in the DRN and some of its projection sites using a selective OX1R antagonist SB-334867 (10 mg/kg, i.p.). A single injection of SB-334867 decreased 5-HT-ir fibers within the anterior cingulate cortex (aCgC); five once-daily administrations of SB-334867 decreased 5-HT-ir not only in the aCgC but also in the DRN, oval bed nucleus of the stria terminalis (ovBNST), nucleus accumbens shell (NAcSh), and periaqueductal gray (PAG). HPLC analysis revealed that five once-daily administrations of SB-334867 did not affect 5-HT turnover any of the five sites, although it increased the levels of both 5-HT and 5-HIAA in the NAcSh. These results together suggest that orexinergic modulation of DRN 5-HT neurons via OX1Rs may be one pathway through which orexin regulates mood and anxiety, as well as perhaps other neurobiological processes.