As a normal physiological process, sleep has recently been shown to facilitate clearance of macromolecular metabolic wastes from the brain via the glymphatic system. The aim of the present study was to investigate pathophysiological roles of astroglial aquaporin 4 (AQP4), a functional regulator of glymphatic clearance, in a mouse model of chronic sleep disruption (SD). Adult AQP4 null mice and wild-type (WT) mice were given 7 days of SD using the improved rotating rod method, and then received behavioral, neuropathological, and neurochemical analyses. Aquaporin 4 deletion resulted in an impairment of glymphatic transport and accumulation of β-amyloid and Tau proteins in the brain following SD. AQP4 null SD mice exhibited severe activation of microglia, neuroinflammation, and synaptic protein loss in the hippocampus, as well as decreased working memory, compared with WT-SD mice. These results demonstrate that AQP4-mediated glymphatic clearance ameliorates brain impairments caused by abnormal accumulation of metabolic wastes following chronic SD, thus serving as a potential target for sleep-related disorders.