A novel transcriptional regulator responding to glucose was designed with a substrate-binding protein (SBP) as a probe towards intracellular sensing system for glucose in mammalian cells. A chimeric protein of an SBP for glucose (GBP) and a LacI-type regulator, LacI (SLCPGL), was designed, constructed and characterized using Escherichia coli recombinant protein. We report that SLCPGL has a glucose-specific binding ability and an operator-sequence specific DNA-binding ability. The loss of its DNA-binding ability in the presence of glucose suggests a role as a transcriptional regulator in vitro. The glucose-dependent gene regulation function of SLCPGL in cells was investigated using mammalian cells co-transfected with SLCPGL and Lac operator-fused luciferase gene constructs. The luciferase activity of the transfected cells increased with the glucose concentration in the medium, showing that the expression of the luciferase gene is regulated by SLCPGL, which can dissociate from DNA in a glucose concentration-dependent manner. Therefore, we demonstrated that SLCPGL functions as a glucose-sensitive transcriptional regulator in mammalian cells. These results reveal the possibility of developing an SBP-based regulator as a probe of intracellular sensing and gene regulation system for mammalian cells in response to a desired ligands depending on the SBP ligand specificity. Biotechnol. Bioeng. 2011; 108:725–733. © 2010 Wiley Periodicals, Inc.