The role of ventral hippocampus (vHipp) astroglial gliotransmission in depression was studied using chronic restraint stress (CRS) and chronic unpredictable mild stress (CUMS) rodent models. CRS increased Cx43 hemichannel activity and extracellular glutamate levels in the vHipp and blocking astroglial Cx43 hemichannel-dependent gliotransmission during CRS prevented the development of depression and glutamate buildup. Moreover, the acute blockade of Cx43 hemichannels induced antidepressant effects in rats previously subjected to CRS or CUMS. This antidepressant effect was prevented by coinjection of glutamate and D-serine. Furthermore, Cx43 hemichannel blockade decreased postsynaptic NMDAR currents in vHipp slices in a glutamate and D-serine-dependent manner. Notably, chronic microinfusion of glutamate and D-serine, L-serine, or the NMDAR agonist NMDA, into the vHipp induced depressive-like symptoms in nonstressed rats. We also identified a small molecule, cacotheline, which blocks Cx43 hemichannels and its systemic administration induced rapid antidepressant effects, preventing stress-induced increases in astroglial Cx43 hemichannel activity and extracellular glutamate in the vHipp, without sedative or locomotor side effects. In conclusion, chronic stress increases Cx43 hemichannel-dependent release of glutamate and D-/L-serine from astrocytes in the vHipp, overactivating postsynaptic NMDARs and triggering depressive-like symptoms. This study highlights the critical role of astroglial gliotransmitter release in chronic stress-induced depression and suggests it can be used as a target for the prevention and treatment of depression.