Stress-related alterations in brain-derived neurotrophic factor (BDNF) expression, a neurotrophin that plays a key role in synaptic plasticity, are believed to contribute to the pathophysiology of depression. Here, we show that in a chronic mild stress (CMS) model of depression the G alpha i1 and G alpha i3 subunits of heterotrimeric G proteins are down-regulated in the hippocampus, a key limbic structure associated with major depressive disorder. We provide evidence that G alpha i1 and G alpha i3 (G alpha i1/3) are required for the activation of TrkB downstream signaling pathways. In mouse embryonic fibroblasts (MEFs) and CNS neurons, G alpha i1/3 knockdown inhibited BDNF-induced tropomyosin-related kinase B (TrkB) endocytosis, adaptor protein activation, and Akt-mTORC1 and Erk-MAPK signaling. Functional studies show that G alpha i1 and G alpha i3 knockdown decreases the number of dendrites and dendritic spines in hippocampal neurons. In vivo, hippocampal G alpha i1/3 knockdown after bilateral microinjection of lentiviral constructs containing G alpha i1 and G alpha i3 shRNA elicited depressive behaviors. Critically, exogenous expression of G alpha i3 in the hippocampus reversed depressive behaviors in CMS mice. Similar results were observed in G alpha i1/G alpha i3 double-knockout mice, which exhibited severe depressive behaviors. These results demonstrate that heterotrimeric G alpha i1 and G alpha i3 proteins are essential for TrkB signaling and that disruption of G alpha i1 or G alpha i3 function could contribute to depressive behaviors.