Correlations amongst the rat glutamatergic system, glia, and depression, as well as the underlying mechanism of astrocyte impairment, as a trigger of depression, were investigated. Rats were submitted to different durations of chronic unpredictable mild stress to induce depressive-like behavior and evaluated by weight change, sucrose preference test, open field test, and novelty suppressed feeding test. High-performance liquid chromatography was employed to detect glutamate content of hippocampal protein expression during Western blot and immunofluorescence. Results showed that 21-day chronic unpredictable mild stress was sufficient for inducing significant depressive-like behavior (reduced body weight and sucrose preference, increased feeding, and immobility time) in a model of depression. Chronic unpredictable mild stress increased the level of hippocampal glutamate, while intervention caused a considerable rise in the expression levels of Bax, caspase 3, and calcium/calmodulin-dependent protein kinase II, accompanied by a down-regulated level of B-cell lymphoma-2. Exposure to this stress model reduced hippocampal glutamate ionotropic receptor N-methyl-D-aspartic acid type subunit 2A, neuronal nuclear protein, and glial fibrillary acidic protein expression levels while it raised the level of ionotropic glutamate receptor N-methyl-D-aspartic acid type subunit 2B level. It is concluded that chronic stress induces excessive glutamate release and overstimulation of N-methyl-D-aspartic acid receptors, followed by astrocytic apoptosis. Also, in depression, calcium overload in astrocytes is attributed to an underlying mechanism of astrocyte impairment.