The duodenal homeobox-1 protein Pdx-1 is one of the regulators for the transcription of the insulin gene. Pdx-1 is a phosphoprotein, and there is increasing evidence for the regulation of some of its functions by phosphorylation. Here, we asked whether protein kinase CK2 might phosphorylate Pdx-1 and how this phosphorylation could be implicated in the functional regulation of Pdx-1. We used fragments of Pdx-1 as well as phosphorylation mutants for experiments with protein kinase CK2. Transactivation was measured by reporter assays using the insulin promoter. Our data showed that Pdx-1 is phosphorylated by protein kinase CK2 at amino acids thr(231) and ser(232), and this phosphorylation was implicated in the regulation of the transcription factor activity of Pdx-1. Furthermore, inhibition of protein kinase CK2 by specific inhibitors led to an elevated release of insulin from pancreatic beta-cells. Thus, these findings identify CK2 as a novel mediator of the insulin metabolism.