Protein 14-3-3e also named as monooxygenase/tryptophan 5-monooxygenase activation protein has known as interactional partner of several transcriptional factors and subsequently regulates their activity. In studying of the role of 14-3-3e protein in the activation of an insulin gene transcription via PDX-I (pancreatic duodenal homeobox-1) factor, RNA interference (RNAi) technology was applied to selectively inhibit its expression in the cells. Two target sequences on the 14-3-3e cDNA were selected to construct siRNA-expressing vectors. Besides that, a reporter vector was generated by inserting the cDNA of 14-3-3e downstream the renilla luciferase gene in the vector. The luciferase assay results showed that one of two constructed siRNA expression vectors has an ability to produce si 14-3-3e and block the expression of its target gene up to 85 percent in the AD293 cell. The expression of si 14-3-3e completely inhibited the over-expression of the exogenous 14-3-3e in the AD293 cell. For endogenous 14-3-3e in insulin-producing cells, si 14-3-3e can block the expression of protein up to 60 percent. In conclusion, the si 14-3-3e expressing vector was generated and its function was demonstrated. This vector could be used to study 14-3-3e roles not only in the insulin gene transcription activation but also in other mechanism of cell.