AbstractCalcium-dependent secretion activator 2 (CAPS2) regulates the trafficking and exocytosis of neuropeptide-containing dense-core vesicles (DCVs). CAPS2 is prominently expressed in the medial habenula (MHb), which is related to depressive behavior; however, how MHb neurons cause depressive symptoms and the role of CAPS2 remains unclear. We hypothesized that dysfunction of MHb CAPS neurons might cause defects in neuropeptide secretion and the activity of monoaminergic centers, resulting in depressive-like behaviors. In this study, we examined (1) CAPS2 expression in the habenula of depression animal models and major depressive disorder patients and (2) the effects of down-regulation of MHb CAPS2 on the animal behaviors, synaptic transmission in the interpeduncular nucleus (IPN), and neuronal activity of monoamine centers. Habenular CAPS2 expression was decreased in the rat chronic restraint stress model, mouse learned helplessness model, and showed tendency to decrease in depression patients who died by suicide. Knockdown of CAPS2 in the mouse habenula evoked despair-like behavior and a reduction of the release of DCVs in the IPN. Neuronal activity of IPN and monoaminergic centers was also reduced. These results implicate MHb CAPS2 as playing a pivotal role in depressive behavior through the regulation of neuropeptide secretion of the MHb-IPN pathway and the activity of monoaminergic centers.