OBJECTIVE: Glucose promotes lipid remodelling in pancreatic β-cells, and this is thought to contribute to the regulation of insulin secretion, but the metabolic pathways and potential signalling intermediates have not been fully elaborated. METHODS: Using mass spectrometry (MS) we quantified changes in approximately 300 lipid metabolites in MIN6 β-cells and isolated mouse islets following 1 h stimulation with glucose. Flux through sphingolipid pathways was also assessed in (3)H-sphinganine-labelled cells using TLC. RESULTS: Glucose specifically activates the conversion of triacylglycerol (TAG) to diacylglycerol (DAG). This leads indirectly to the formation of 18:1 monoacylglycerol (MAG), via degradation of saturated/monounsaturated DAG species, such as 16:0_18:1 DAG, which are the most abundant, immediate products of glucose-stimulated TAG hydrolysis. However, 16:0-containing, di-saturated DAG species are a better direct marker of TAG hydrolysis since, unlike the 18:1-containing DAGs, they are predominately formed via this route. Using multiple reaction monitoring, we confirmed that in islets under basal conditions, 18:1 MAG is the most abundant species. We further demonstrated a novel site of glucose to enhance the conversion of ceramide to sphingomyelin (SM) and galactosylceramide (GalCer). Flux and product:precursor analyses suggest regulation of the enzyme SM synthase, which would constitute a separate mechanism for localized generation of DAG in response to glucose. Phosphatidylcholine (PC) plasmalogen (P) species, specifically those containing 20:4, 22:5 and 22:6 side chains, were also diminished in the presence of glucose, whereas the more abundant phosphatidylethanolamine plasmalogens were unchanged. CONCLUSION: Our results highlight 18:1 MAG, GalCer, PC(P) and DAG/SM as potential contributors to metabolic stimulus-secretion coupling.