Impairment of amyloid-beta (A beta) clearance leads to A beta accumulation in the brain during the development of Alzheimer's disease (AD). Strategies that can restore or improve the clearance function hold great promise in delaying or preventing the onset of AD. Here, we show that n-3 polyunsaturated fatty acids (PUFAs), by use of fat-1 transgenic mice and oral administration of fish oil, significantly promote interstitial A beta clearance from the brain and resist A beta injury. Such beneficial effects were abolished in Aqp4-knockout mice, suggesting that the AQP4-dependent glymphatic system is actively involved in the promoting the effects of n-3 PUFAs on the clearance of extracellular A beta. Imaging on clarified brain tissues clearly displayed that n-3 PUFAs markedly inhibit the activation of astrocytes and protect the AQP4 polarization in the affected brain region after A beta injection. The results of the present study prove a novel mechanism by which n-3 PUFAs exert protective roles in reducing A beta accumulation via mediating the glymphatic system function.-Ren, H., Luo, C., Feng, Y., Yao, X., Shi, Z., Liang, F., Kang, J. X., Wan, J.-B., Pei, Z., Su, H. Omega-3 polyunsaturated fatty acids promote amyloid-b clearance from the brain through mediating the function of the glymphatic system.