Abstract: Background: Mood disorders such as depression and metabolic diseases like obesity and diabetes have significant comorbidities; furthermore, each disease increases the risk of the other one. Continuing use of antidepressant medication is associated with an elevated risk of type 2 diabetes and weight gain. On the other hand, antiobesity drugs are found to have varying neuropsychiatric adverse event profiles. Therefore, there is an urgent need to develop more effective and safer therapies for mood disorders and metabolic diseases with minimal side effects. Both brain derived neurotrophic factor (BDNF) and serotonin (5-HT) can regulate moods in the limbic system and metabolism in the hypothalamus, respectively. The dorsal raphe nucleus (DR) in the midbrain contains the largest number of serotonin-expressing neurons as well as other neurons expressing various neurotransmitters. Both serotonergic and nonserotonergic DR neurons send projections widely to the forebrain including the limbic system and the hypothalamus. This study aimed to investigate whether BDNF gene transfer in DR neurons can improve moods and metabolism. Methods: Recombinant adeno-associated virus (AAV) was microinjected into DR to achieve BDNF gene transfer. Chronic unpredictable mild stress (CUMS) model of depression in C57BL/6 mice was established to investigate the role of DR BDNF gene transfer in depression. Forced swimming test (FST) and sucrose consumption test (SCT) were used to examine the depressive-like behaviors. Open field test (OPT) and elevated zero maze (EZM) were used to examine the anxiety-like behaviors. Diet-induced obesity (DIO) and leptin receptor mutant db/db mice were employed to investigate the role of DR BDNF gene transfer in regulating metabolism. Metabolic profiling included body weight, food intake, fat mass, serum factors, energy expenditure, glucose tolerance and insulin sensitivity. Quantitative RT-PCR was performed to determine expression changes of several key genes in DR, hypothalamus, liver and fat tissues. Tph2 conditional knockout mice were used to examine whether 5-HT is essential for BDNF in improving metabolism. In addition, chemogenetic activation of DR neurons via AAV-mediated hM3Dq expression was also performed to examine whether there is a similar effect as BDNF. Results: FST and SCT showed significant anti-depressant effects of DR BDNF gene transfer in the CUMS model, meanwhile, OPT and EZM showed significant anxiolytic effects. BDNF gene transfer in DR also markedly ameliorated obesity and hyperglycemia in DIO and db/db mice, which was revealed by lightened body weight, decreased food intake, increased energy expenditure, improved glucose tolerance and insulin sensitivity, reduced levels of serum factors like insulin and leptin. The mRNA expression of genes related to 5-HT signaling such as tph2 and vmat2 was significantly increased in DR after BDNF gene transfer. Expression levels of multiple genes regulating energy balance in the hypothalamus, liver, and fat tissues also changed. Conditional knockout of tph2, encoding the rate-limiting enzyme of 5-HT synthesis, did not block the effects of DR BDNF gene transfer in improving metabolism. Chemogenetic activation of DR neurons also had the effects of improving metabolism in DIO mice. Conclusions: BDNF gene transfer in DR of mice improves moods and metabolism. This study surmounts the “hypothalamocentric” paradigm dominating in metabolism research by connecting metabolism and moods via dorsal raphe.