Aquaporin-4 (AQP4) water channels are concentrated in astrocytic endfoot membranes at the brain–blood and brain–cerebrospinal fluid interfaces. The mechanisms underpinning the polarized distribution of AQP4 are poorly understood. Here we tested the hypothesis that pericytes regulate AQP4 anchoring to perivascular astrocytic endfoot membranes. AQP4 immunofluorescence of brain sections obtained from novel transgenic double reporter mice expressing enhanced green fluorescent protein (eGFP) in astrocytes and Discoma Red (DsRed) in pericytes revealed strong AQP4 signal in astrocytic processes adjacent to pericytes. Quantitative immunogold analysis of C57BL/6 mice showed that the AQP4 expression was higher in endfoot membranes abutting pericytes than in those facing endothelial cells. Similar findings were made for α-syntrophin, a member of the dystrophin-associated protein complex (DAPC). The enrichment of α-syntrophin in membranes ensheathing pericytes persisted after Aqp4 gene deletion. Our data support the concept that pericytes regulate AQP4 polarization.