Acyl-CoA-binding protein (ACBP) acts as an acyl-CoA pool former, transporter, and regulator of gene transcription in vitro. We created a transgenic rat line overexpressing ACBP, as the physiological relevance of ACBP in lipid metabolism is unclear. Transgenic rats revealed increased levels of ACBP and significantly elevated acyl-CoA tissue levels while there was no effect on plasma triglyceride, cholesterol, or serum-free fatty acid levels. Metabolic regulators like peroxisome proliferator-activated receptors (PPARgamma, PPARdelta) and sterol regulatory element-binding protein-1 (SREBP-1) messenger RNA levels were significantly reduced (by 23-82%) in liver and adipose tissue of fed transgenic rats, whereas adenosine monophosphate-activated protein kinase (AMPK) protein levels were increased (by 60%). Fasting abolished PPAR downregulation in liver and caused an upregulation in adipose tissue. Administration of AMPK inhibitor reversed SREBP-1 but did not affect PPAR regulation. In conclusion, ACBP acts as an acyl-CoA pool former in transgenic rats and regulates lipid metabolism via SREBP-1 and PPAR regulation. Reduction of SREBP-1 is mediated via increased AMPK levels, whereas regulation of PPARs seems to be mediated by an AMPK-independent mechanism. ACBP itself is a target gene for both transcription factors demonstrating important feedback loops.