Aquaporin-4 (AQP4), a key molecule for maintaining water homeostasis in the central nervous system, is expressed in adult neural stem cells (ANSCs) as well as astrocytes. Neural stem cells give rise to new hippocampal neurons throughout adulthood, and defects in neurogenesis may predispose an individual to depression. Nevertheless, the role of AQP4 in adult hippocampal neurogenesis and chronic mild stress (CMS)-induced depression remains unknown. We herein report that AQP4 knockout disrupted 4-week fluoxetine (10 mg/kg per day i.p) treatment-induced enhancement of adult mouse hippocampal neurogenesis as well as behavioral improvement under both basal condition and CMS-evoked depressive state. Meanwhile, AQP4 knockout abolished fluoxetine-induced enhancement of hippocampal cyclic AMP-responsive element binding protein (CREB) phosphorylation. The CMS procedure inhibited hippocampal protein kinase A (PKA) activity, extracellular signal-regulated kinases (ERK1/2), and calcium/calmodulin-dependent protein kinase IV (CaMKIV) phosphorylation in AQP4(+/+) and AQP4(-/-) mice. Fluoxetine treatment could reverse CMS-induced inhibition of PKA activity and ERK1/2 phosphorylation in both genotypes. However, fluoxetine restored CMS-induced inhibition of hippocampal CaMKIV phosphorylation in AQP4(+/+) mice but failed in AQP4(-/-) mice. Notably, CMS procedure significantly increased the hippocampal AQP4 expression, which was reversed by 4-week fluoxetine treatment. Further investigation showed AQP4 knockout inhibited the proliferation of cultured ANSCs and eliminated the pro-proliferative effect of fluoxetine in vitro. Collectively, these findings suggest that AQP4 is required for the antidepressive action of fluoxetine via regulating adult hippocampal neurogenesis.