Chronic sleep restriction (SR) impairs the glymphatic clearance of macromolecular toxic metabolites, which is associated with the loss of perivascular polarization of aquaporin-4 (AQP4). Melatonin (Mel) has been shown to maintain the circadian rhythm of AQP4 polarization. However, the role of AQP4 polarization in Mel's protective effects against SR-induced brain dysfunction remains unclear. In the present study, using a modified rotating rod SR mouse model, we demonstrated the time-dependent effect of SR on short-term memory deficits and AQP4 mislocalization in the hippocampus. Subsequent experiments characterized the dose-dependent pattern of Mel ameliorating SR-induced impairments of cognitive function and AQP4 polarity. Mel's treatment enhanced glymphatic transport in SR mice, as revealed by cerebrospinal tracer experiments, and reduced hippocampal amyloid-beta and phosphorylated tau levels. Additionally, Mel significantly decreased glial cell activation, pro-inflammatory cytokine production, and synaptic protein loss in the hippocampus of SR mice. However, in AQP4 knockout mice, Mel's protective effects against SR-induced pathophysiological alterations described above were largely abolished. Mechanistically, Mel activated the vitamin D receptor and then upregulated expression of DTNA (Dystrobrevin Alpha), a key component of the dystrophin-associated complex, which in turn restored AQP4 polarization during chronic SR conditions. This finding indicates that AQP4-mediated lymphatic clearance is necessary for Mel to combat chronic SR-induced brain impairment.