Sphingolipids (SLs), such as ceramide, glucosylceramide (GlcCer), and sphingomyelin, play important roles in the normal development/functions of the brain and peripheral tissues. Disruption of SL homeostasis in cells/organelles, specifically up-regulation of ceramide, is involved in multiple diseases including Alzheimer’s disease (AD). One of the pathological features of AD is aggregates of amyloid beta (Aβ) peptides, and SLs regulate both the formation/aggregation of Aβ and Aβ-induced cellular responses. Up-regulation of ceramide levels via de novo and salvage synthesis pathways is reported in Aβ-treated cells and brains with AD; however, the effects of Aβ on ceramide decomposition pathways have not been elucidated. Thus, we investigated the effects of the 25–35-amino acid Aβ peptide (Aβ25–35), the fundamental cytotoxic domain of Aβ, on SL metabolism in cells treated with the fluorescent nitrobenzo-2-oxa-1,3-diazole-labeled C6-ceramide (NBD-ceramide). Aβ25–35 treatment reduced the formation of NBD-GlcCer mediated by GlcCer synthase (GCS) without affecting the formation of NBD-sphingomyelin or NBD-ceramide-1-phosphate, and reduced cell viability. Aβ25–35-induced responses decreased in cells treated with D609, a putative inhibitor of sphingomyelin synthases. Aβ25–35-induced cytotoxicity significantly increased in GCS-knockout cells and pharmacological inhibition of GCS alone demonstrated cytotoxicity. Our study revealed that Aβ25–35-induced cytotoxicity is at least partially mediated by the inhibition of GCS activity.