The transcriptional repressor RE1 silencer transcription factor (NRSF/REST) is an important factor that restricts some neuronal traits in neurons. Since these traits are also present in pancreatic islet cells, NRSF-regulated genes involved in islet function are searched. A NRSE-like motif was analysed in human insulin promoter. The role of NRSE was evaluated by generating a model of insulin-secreting cells that firmly express NRSF. The presence of NRSF led to a decrease in activity of human insulin promoter by stable or transient transfection with human insulin-promoter luciferase. The predicted NRSE-like motif also confers NRSF-dependent transcriptional repression in the context of a surrogate gene promoter. Specific binding activity of NRSF/REST to the NRSE-like motif was confirmed by EMSA. Moreover, the binding activity is competed by consensus NRSE sequence. These data showed that human insulin promoter is regulated by the transcriptional repressor NRSF/REST via the NRSE-like motif.