Hypercholesteremia results in cognitive decline, while higher intakes of marine n-3 polyunsaturated fatty acids (PUFA) can ameliorate mild cognitive impairment (MCI). However, whether individual n-3 PUFA, notably docosahexaenoic acid (DHA, C22:6n-3) and eicosapentaenoic acid (EPA, C20:5n-3) have different efficiency to ameliorate MCI remain ambiguous. This study aimed to investigate the underlying mechanism why DHA had superior functions to ameliorate MCI. Here, a case-control study with 1:2 case-to-control ratio was implemented. It showed that serum ceramide concentrations were significantly higher in MCI patients compared with healthy controls, and were negatively associated with DHA composition in erythrocyte phospholipids. The MCI model was established with male C57BL/6J wild-type mice fed a high-fat and high-cholesterol diet (HFD), and administration of DHA led to ameliorated cognitive behavior abnormalities and neuronal apoptosis. The improvement of MCI phenotype was further recapitulated in low-density lipoprotein receptor knockout (LDLR−/−) mice treated with DHA. Mechanistically, DHA could remodel composition of phospholipid membranes, leading to reduced endocannabinoid ligands and inhibited cannabinoid receptor 1 (CB1). The restrained endocannabinoid system was bound up with inhibited ceramide-induced apoptosis of neurons. This work reveals a novel mechanism, namely CB1-ceramide axis, through which DHA exhibits an alleviation in HFD-induced MCI.