The lateral habenula (LHb) has recently emerged as a key brain region in the pathophysiology of depression. However, the molecular mechanism by which LHb becomes hyperactive in depression remains unknown. Through a quantitative proteomic screen, we found that expression of the β form of calcium/calmodulin-dependent protein kinase type II (βCaMΚΙΙ) was significantly up-regulated in the LHb of animal models of depression and down-regulated by antidepressants. Increasing β-, but not α-, CaMKII in the LHb strongly enhanced the synaptic efficacy and spike output of LHb neurons and was sufficient to produce profound depressive symptoms, including anhedonia and behavioral despair. Down-regulation of βCaMKII levels, blocking its activity or its target molecule the glutamate receptor GluR1 reversed the depressive symptoms. These results identify βCaMKII as a powerful regulator of LHb neuron function and a key molecular determinant of depression.