Serum response factor (SRF) is an essential regulator of myogenic and neurogenic genes and the ubiquitously expressed immediate-early genes. The purpose of this study is to determine SRF expression pattern in murine pancreas and examine the role of SRF in pancreatic gene expression. Immunohistochemical analysis of wild-type pancreas and LacZ staining of pancreas from SRF LacZ knock-in animals showed that SRF expression is restricted to β cells. SRF bound to the rat insulin promoter II (RIP II) serum response element, an element conserved in both rat I and murine I and II insulin promoters. SRF activated RIP II, and SRF binding to RIP II and the exon 5-encoded 64-aa subdomain of SRF was required for this activation. Transient or stable knockdown of SRF leads to down-regulation of insulin gene expression, suggesting that SRF is required for insulin gene expression. Further, SRF physically interacted with the pancreas and duodenum homeobox-1 (Pdx-1) and synergistically activated RIP II. Elevated glucose concentration down-regulated SRF binding to RIP II SRE, and this down-regulation was associated with decreased RIP II activity and increased SRF phosphorylation on serine 103. Together, our results demonstrate that SRF is a glucose concentration-sensitive regulator of insulin gene expression.