The transcriptional repressor RE1 silencer transcription factor (NRSF/REST) is an important factor that restricts some neuronal traits in neurons. It has been confirmed that the insulin gene was regulated by NRSF via NRSE. Here, the relationship between NRSF and insulin gene was investigated by means of RNA interference (RNAi). The expression of NRSF was down-regulated by lentiviral RNA interference vector, and the interference efficiency is 56% (n = 6, P < 0.01). When the expression of NRSF was down-regulated, genes regulated by NRSF, such as SCG10, BDNF, pax4 and insulin, began to express in HeLa cells. The human insulin promoter activity was up-regulated 2.4-fold after RNA interference (n = 3, P < 0.01), confirmed by dual-luciferase assay.