Changes in extracellular glucose levels regulate the expression of the immediate early response gene and zinc finger transcription factor early growth response (Egr)-1 in insulin-producing pancreatic cells, but key target genes of Egr-1 in the endocrine pancreas have not been identified. We found that overexpression of Egr-1 in clonal (INS-1) cells increased the transcriptional activation of the rat insulin I promoter. In contrast, reductions of Egr-1 expression levels or function with the introduction of either small interfering RNA targeted to Egr-1 (siEgr-1) or of a dominant-negative form of Egr-1 decreased insulin promoter activation, and siEgr-1 suppressed insulin gene expression. Egr-1 did not directly interact with insulin promoter sequences and mutagenesis of a potential G-box recognition sequence for Egr-1 did not impair the Egr-1-responsiveness of the insulin promoter, suggesting that regulation of insulin gene expression by Egr-1 likely is mediated through additional transcription factors. Overexpression of Egr-1 increased, and reduction of Egr-1 expression decreased, the transcriptional activation of the glucose-responsive FarFlat minienhancer within the rat insulin I promoter, despite the absence of demonstrable Egr-1 binding activity to FarFlat sequences. Notably, augmenting Egr-1 expression levels in insulin-producing cells increased the mRNA and protein expression levels of pancreas duodenum homeobox-1 (PDX-1), a major transcriptional regulator of glucose-responsive activation of the insulin gene. Increasing Egr-1 expression levels enhanced PDX-1 binding to insulin promoter sequences, while mutagenesis of PDX-1-binding sites reduced the capacity of Egr-1 to activate the insulin promoter. We propose that changes in Egr-1 expression levels, in response to extracellular signals including glucose, can regulate PDX-1 expression and insulin production in pancreatic cells.