The glymphatic system transports cerebrospinal fluid (CSF) into the brain via arterial perivascular spaces and removes interstitial fluid from the brain along perivenous spaces and white matter tracts. This directional fluid flow supports the clearance of metabolic wastes produced by the brain. Glymphatic fluid transport is facilitated by aquaporin-4 (AQP4) water channels, which are enriched in the astrocytic vascular endfeet comprising the outer boundary of the perivascular space. Yet, prior studies of AQP4 function have relied on genetic models, or correlated altered AQP4 expression with glymphatic flow in disease states. Herein, we sought to pharmacologically manipulate AQP4 function with the inhibitor AER-271 to assess the contribution of AQP4 to glymphatic fluid transport in mouse brain. Administration of AER-271 inhibited glymphatic influx as measured by CSF tracer infused into the cisterna magna and inhibited increases in the interstitial fluid volume as measured by diffusion-weighted MRI. Furthermore, AER-271 inhibited glymphatic efflux as assessed by an in vivo clearance assay. Importantly, AER-271 did not affect AQP4 localization to the astrocytic endfeet, nor have any effect in AQP4 deficient mice. Since acute pharmacological inhibition of AQP4 directly decreased glymphatic flow in wild-type but not in AQP4 deficient mice, we foresee AER-271 as a new tool for manipulation of the glymphatic system in rodent brain.