Major depressive disorder (MDD) is a devastating mental disorder that affects up to 17% of the population worldwide. Although brain-wide network-level abnormalities in MDD patients via resting-state functional magnetic resonance imaging (rsfMRI) exist, the mechanisms underlying these network changes are unknown, despite their immense potential for depression diagnosis and management. Here, we show that the astrocytic calcium-deficient mice, inositol 1,4,5-trisphosphate-type-2 receptor knockout mice ( Itpr2 ^−/− mice), display abnormal rsfMRI functional connectivity (rsFC) in depression-related networks, especially decreased rsFC in medial prefrontal cortex (mPFC)–related pathways. We further uncover rsFC decreases in MDD patients highly consistent with those of Itpr2 ^−/− mice, especially in mPFC-related pathways. Optogenetic activation of mPFC astrocytes partially enhances rsFC in depression-related networks in both Itpr2 ^−/− and wild-type mice. Optogenetic activation of the mPFC neurons or mPFC-striatum pathway rescues disrupted rsFC and depressive-like behaviors in Itpr2 ^−/− mice. Our results identify the previously unknown role of astrocyte dysfunction in driving rsFC abnormalities in depression.