Depression is the single largest contributor to the burden of disease, yet the current antidepressant medications are limited by high non-responsiveness and significant side effects. The lateral septum (LS) is thought to control of depression, however, the cellular and circuit substrates are largely unknown. Here, we identified a subpopulation of LS GABAergic adenosine A2A receptors (A2AR)-positive neurons mediating depression via direct projects to the lateral habenula (LHb) and the hypothalamus. Activation of A2AR in the LS augmented the spiking frequency of A2AR-positive neurons leading to a decreased activation of surrounding neurons. Accordingly, modulation of LS-A2AR-positive neurons activity via optogenetic stimulation formatted depressive-like phenotype and the optogenetic activation of LS-A2AR-positive neurons projection terminals to the LHb or the hypothalamus, phenocopied depressive behaviors. Moreover, we shown a selective upregulation of A2AR in the LS in two mouse models of repeated stress-induced depression and in postmortem brains of suicide completers suffered from depression disorder, and the bi-directional manipulation of LS-A2AR activity demonstrated that LS-A2ARs are necessary and sufficient to trigger depressive phenotypes. This identification that aberrantly increased A2AR signaling in the LS is a critical upstream regulator of repeated stress-induced depressive-like behaviors provides a neurophysiological and circuit-based justification of the antidepressant potential of A2AR antagonists, prompting their clinical translation.