Both peroxisome proliferator-activated receptor-alpha (PPAR) and pancreatic/duodenal homeobox-1 (PDX-1) have been reported to be associated with glucose-stimulated insulin secretion (GSIS), but the relationship between PPAR and PDX-1 is not yet fully understood. In the present study, we tested the hypothesis that PPAR regulates the expression of PDX-1 in -cells. Isolated pancreatic islets from Wistar rats and INS-1 pancreatic -cells were cultured in media supplemented with and without 0.2 mM or 0.4 mM palmitate, and treated with and without a PPAR agonist (fenofibrate) or PPAR antagonist (MK886). Results indicated that treatment with fenofibrate significantly enhanced PPAR mRNA and protein expression in cells cultured with elevated palmitate concentrations compared to cells that did not receive fenofibrate treatment. In turn, this enhanced expression led to an increase in PDX-1 mRNA and nuclear protein, as well as DNA binding activity of PDX-1 with the insulin promoter. Accordingly, the expression of the PDX-1 downstream targets, insulin and glucose transporter-2, increased, resulting in increased intracellular insulin content and GSIS. Treatment with MK886 inhibited expression of PPAR, blocking PPAR-regulated PDX-1 expression, and the downstream transcription events of PDX-1. EMSA revealed that nuclear protein might bind with the PPRE sequence located in the PDX-1 promoter. Collectively, these results demonstrate a regulatory relationship between PPAR and PDX-1 in INS-1 cells. Furthermore, PPAR activation potentiates GSIS under elevated palmitate conditions possibly via up-regulation of PDX-1. Our findings have potential clinical implications for the use of PPAR agonists in the treatment of type 2 diabetes.