Transcriptional regulation of the insulin gene plays a critical role in maintenance of pancreatic beta cell function in response to various stimuli. Here, we used INS-1 cells to test the hypothesis that PGC-1alpha regulates human insulin gene transcription by modulating glucocorticoid (GR) binding to the insulin gene promoter. Analysis of the human insulin promoter region revealed that the suppressive region regulated by GR and PGC-1alpha is localized from -362 to -257 bp. To locate the GR binding site in the human insulin promoter region, EMSAs were performed with candidate GR binding sequences and confirmed that a palindromic region (Palin, -284 to -274 bp) specifically interacts with GR. We also found that the Palin-binding activity of GR is increased in the presence of PGC-1alpha. These findings suggest that PGC-1alpha elevates the binding of GR to Palin and thereby enhances the GR-mediated inhibition of human insulin transcription.