NKX3.1 is a prostate-specific homeobox gene related to prostate development and prostate cancer. In this work, we aimed to identify precisely the functional cis-element in the 197 bp region (from -1032 to -836 bp) of the NKX3.1 promoter (from -1032 to +8 bp), which was previously identified to present positive regulatory activity on NKX3.1 expression, by deletion mutagenesis analysis and electrophoretic mobility shift assay (EMSA). A 16 bp positive cis-element located between -920 and -905 bp upstream of the NKX3.1 gene was identified by deletion mutation analysis and proved to be a functional positive cis-element by EMSA. It will be important to further study the functions and regulatory mechanisms of this positive cis-element in NKX3.1 gene expression.