Peroxisome proliferator-activated receptor-gamma co-activator-1 (PGC-1) alpha and -beta play pivotal roles in the regulation of intermediary metabolism. We have previously shown that PGC-1alpha-mediated upregulation of beta-cell sterol element binding protein (SREBP) gene expression impairs insulin secretion via increased transcription of uncoupling protein 2 (UCP2). PGC-1beta, in contrast to PGC-1alpha, directly binds to and acts as a co-activator of SREBPs and the forkhead transcription factor 2A (FOXA2) involved in pancreas development and function. To address a possible role of PGC-1beta in beta-cell function, we determined islet gene expression levels of PGC-1alpha, PGC-1beta, SREBPs, FOXA2, FOXO1, UCP2 as well as granuphilin, a critical component of the insulin secretory machinery, in Zucker diabetic fatty rats (ZDF). In comparison to controls, mRNA levels of all genes studied except for FOXA2 and FOXO1 were increased in islets of obese, fa/fa ZDF rats. The transcriptional activities of the UCP2 and granuphilin promoters were assessed in INS-1E cells in response to PGC-1beta overexpression and small interference RNA (siRNA)-mediated gene silencing. PGC-1beta as well as SREBP-1c and -2 increased transcription from the UCP2 promoter in INS-1E cells. Transient transfection of PGC-1beta-specific siRNAs significantly decreased SREBP-2-mediated transcriptional activation of the UCP2 gene. Furthermore PGC-1beta, SREBP-1c, and FOXA2 overexpression augmented granuphilin promoter activity, whereas siRNA-mediated gene knockdown of PGC-1beta reduced the effects of SREBP-1c and FOXA2 on granuphilin gene transcription and significantly increased glucose-stimulated insulin release from INS-1E cells. Our results support a role of PGC-1beta in the regulation of insulin secretion via upregulation of UCP2 and granuphilin gene expression.