Tight regulation of fatty acid metabolism in pancreatic β-cells is important for β-cell viability and function. Chronic exposure to elevated concentrations of fatty acid is associated with β-cell lipotoxicity. Glucose is known to repress fatty acid oxidation and hence to augment the toxicity of fatty acids. The peroxisome proliferator activated receptor α (PPARα) is a key activator of genes involved in β-cell fatty acid oxidation, and transcription of the PPARα gene has been shown to be repressed by increasing concentrations of glucose in β-cells. However, the mechanism underlying this transcriptional repression by glucose remains unclear. Here we report that glucose-induced repression of PPARα gene expression in INS-1E cells is independent of β-cell excitation and insulin secretion but requires activation of protein phosphatase 2A in a process involving inactivation of the AMP-activated protein kinase (AMPK). Pharmacological activation of AMPK at high glucose concentrations interferes with glucose repression of PPARα and PPARα target genes in INS-1E cells as well as in rat islets. Specific knock-down of the catalytic AMPK-subunit AMPKα2 but not AMPKα1 using RNAi suppressed PPARα expression, thereby mimicking the effect of glucose. These results indicate that activation of protein phosphatase 2A and subsequent inactivation of AMPK is necessary for glucose repression of PPARα expression in pancreatic β-cells.