The blood-brain barrier (BBB) is a tightly regulated structure that protect the central nervous system (CNS). BBB impairment is implicated in several brain disorders, such as Alzheimer’s disease. Aquaporin-4 (AQP4) water channels are critically involved in regulating brain water transport across the BBB. We have recently developed a diffusion-weighted pCASL (DW-pCASL) technique allowing measurement of water permeability across the BBB. In this study, we implemented and optimized DW-pCASL in nonhuman primates. We directly validated that the underlying signal mechanism of DW-pCASL is related to AQP4-mediated BBB water exchange using a pharmacological challenge.