Inhibition of GSL (glycosphingolipid) synthesis reduces Aβ (amyloid β-peptide) production in vitro. Previous studies indicate that GCS (glucosylceramide synthase) inhibitors modulate phosphorylation of ERK1/2 (extracellular-signal-regulated kinase 1/2) and that the ERK pathway may regulate some aspects of Aβ production. It is not clear whether there is a causative relationship linking GSL synthesis inhibition, ERK phosphorylation and Aβ production. In the present study, we treated CHO cells (Chinese-hamster ovary cells) and SH-SY5Y neuroblastoma cells, that both constitutively express human wild-type APP (amyloid precursor protein) and process this to produce Aβ, with GSL-modulating agents to explore this relationship. We found that three related ceramide analogue GSL inhibitors, based on the PDMP (D-threo-1-phenyl-2-decanoylamino- 3-morpholino-1-propanol) structure, reduced cellular Aβ production and in all cases this was correlated with inhibition of pERK (phosphorylated ERK) formation. Importantly, the L-threo enantiomers of these compounds (that are inferior GSL synthesis inhibitors compared with the D-threo-enantiomers) also reduced ERK phosphorylation to a similar extent without altering Aβ production. Inhibition of ERK activation using either PD98059 [2-(2-amino-3-methoxyphenyl)-4H- 1-benzopyran-4-one] or U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene) had no impact on Aβ production, and knockdown of endogenous GCS using small interfering RNA reduced cellular GSL levels without suppressing Aβ production or pERK formation. Our data suggest that the alteration in pERK levels following treatment with these ceramide analogues is not the principal mechanism involved in the inhibition of Aβ generation and that the ERK signalling pathway does not play a crucial role in processing APP through the amyloidogenic pathway.