The present study analyzed the amelioration effect and mechanism of two kinds of astaxanthin (AST), including free-AST (F-AST) and docosahexaenoic acid-acylated AST monoester (AST-DHA), on ganglioside (GLS) metabolism in the cortex of APP/PS1 mice using the LC-MS strategy in combination with molecular biology. Water maze and immunohistochemical experiments demonstrated that AST significantly improved the cognitive level of APP/PS1 mice and reduced Aβ deposition in the cortex. After the dietary intake of AST, the composition and level of 84 GLS molecular species in the mouse cortex were determined using the LC-MS strategy. The results showed that the total GLS was reduced, most complex GLS was decreased, and simple GLS (GM3 and GM1a) was increased in the APP/PS1 mouse cortex. Notably, F-AST mainly regulated complex GLS (p < 0.001), whereas AST-DHA primarily reacted with simple GLS (p < 0.001). OAc-GQ1a(38:1), OAc-GQ1a(36:1), GD1a(36:1), and GM3(38:1) decreased 3.73, 2.31, and 2.29-fold and increased 3.54-fold, respectively, and were identified as potential AD biomarkers in the cortices of APP/PS1 mice. Additionally, the AST diet significantly upregulated the mRNA expression of GLS synthesizing genes (st3gal5, st8sia1, b3galt4, st3fal2, and soat) and siae (p < 0.05) and down-regulated that of the GLS catabolizing gene hexa (p < 0.01). In conclusion, improving GLS homeostasis in the AD mouse cortex might be a critical pathway to explain the AD-preventing effect of AST.