Glucose uptake and metabolism are linked to microvascular blood flow and cellular swelling events, which are altered during obesity and can be quantified using magnetic resonance imaging (MRI). Aquaporin-4 (AQP4), the most abundant water-transporting transmembrane protein in the central nervous system, facilitates glucose transport and metabolism-derived water influx. However, its significance and regulatory capacity remain largely unknown. To better understand these processes, we acquired sequential diffusion tensor and T2*-weighted images of the brains of obese and non-obese mice, both before administering an AQP4 inhibitor and after a subsequent glucose challenge. We then subjected the resulting variables to principal component and linear mixed model analyses to assess the influence of diet, sex, administration of the inhibitor, and brain region on the data. Our findings indicate that AQP4-inhibited mice exhibit MRI values consistent with reduced microvascular blood flow and region-specific inhibition of glucose-induced cell swelling during obesity, highlighting a key role for AQP4 in glucose uptake and metabolism. Additionally, we observed that, prior to any experimental manipulation, obese mice displayed MRI signs of lower hippocampal blood flow and cerebral cellular anisotropy compared to controls, in agreement with vascular alterations and reactive gliosis processes.