Serotonin (5-HT) in the central nervous system regulates a variety of biological functions, from the basic homeostatic control to higher brain functions, by acting on fourteen known receptor subtypes. However, it is still usually unclear which receptor subtype is responsible for a specific function due to the lack of highly selective ligands for most of these receptors. Although 5-HT receptor knockout mice are useful, the brain-wide distribution of various receptors makes it difficult to dissect receptor functions in specific and brain regions and cell types. Recent advances in CRISPR/Cas9-mediated in vivo genome editing technology may overcome this problem. In this study, we constructed a viral vector expressing a single guide (sg)RNA targeting Htr1a (sgHtr1a) and Cre recombinase under the control of a neuron-specific promoter. Injection of the viral vector into the dorsal raphe nucleus (DRN) of Cre-dependent Cas9 knock-in mice induced Cre-dependent Cas9 expression mainly in DRN serotonin and GABA neurons. Mismatch cleavage assay and Sanger sequencing showed insertion or deletion formation at the target site. 5-HT1A receptor agonist-induced hypothermia was attenuated and antidepressant effect of a selective serotonin reuptake inhibitor (SSRI) was enhanced by microinjection of the viral vector expressing sgHtr1a into the DRN of Cre-dependent Cas9 knock-in mice. These results suggest that this in vivo CRISPR/Cas9-mediated 5-HT receptor gene knockout strategy provides a reliable and low-cost method for elucidating 5-HT receptor functions in specific cell types and brain regions. Further, we demonstrate that the neuronal 5-HT1A receptor in the DRN regulates body temperature and antidepressant effect of SSRI.