Impairment of glucose-stimulated insulin secretion (GSIS) caused by glucolipotoxicity is an essential feature in type 2 diabetes mellitus (T2DM). Palmitate and eicosapentaenoate (EPA), because of their lipotoxicity and protection effect, were found to impair or restore the GSIS in beta cells. Furthermore, palmitate was found to up-regulate the expression level of sterol regulatory element-binding protein (SREBP)-1c and down-regulate the levels of pancreatic and duodenal homeobox (Pdx)-1 and glucagon-like peptide (GLP)-1 receptor (GLP-1R) in INS-1 cells. To investigate the underlying mechanism, the lentiviral system was used to knock-down or over-express SREBP-1c and Pdx-1, respectively. It was found that palmitate failed to suppress the expression of Pdx-1 and GLP-1R in SREBP-1c-deficient INS-1 cells. Moreover, down-regulation of Pdx-1 could cause the low expression of GLP-1R with/without palmitate treatment. Additionally, either SREBP-1c down-regulation or Pdx-1 over-expression could partially alleviate palmitate-induced GSIS impairment. These results suggested that sequent SREBP-1c-Pdx-1-GLP-1R signal pathway was involved in the palmitate-caused GSIS impairment in beta cells. J. Cell. Biochem. 111: 634642, 2010. (c) 2010 Wiley-Liss, Inc.