In the present study, a functional neuron restrictive silencer element (NRSE) was initially identified in the 5' flanking region (- 83 to - 67, relative to ATG) of human secretin receptor (hSCTR) gene by promoter assays coupled with scanning mutation analyses. The interaction of neuron restrictive silencer factor (NRSF) with this motif was later indicated via gel mobility shift and ChIP assays. The silencing activity of NRSF was confirmed by over-expression and also by shRNA knock-down of endogenous NRSF. These studies showed an inverse relationship between the expression levels of NRSF and hSCTR in the cells. As hSCTR gene was previously shown to be controlled by two GC-boxes which are regulated by the ratio of Sp1 to Sp3, in the present study, the functional interactions of NRSF and Sp proteins to regulate hSCTR gene was investigated. By co-immunoprecipitation assays, we found that NRSF could be co-precipitated with Sp1 as well as Sp3 in PANC-1 cells. Interestingly, co-expressions of these factors showed that NRSF could suppress Sp1-mediated, but not Sp3-mediated, transactivation of hSCTR. Taken together, we propose here that the down-regulatory effects of NRSF on hSCTR gene expression are mediated via its suppression on Sp1-mediated transactivation. © 2012 Elsevier B.V.