We previously demonstrated that the VIL2 −87/+134 region exhibited promoter activity in some human cells, and a region further upstream of this promoter might contain an enhancer. However, the properties and location of this VIL2 enhancer remain unclear. In this study, we cloned the VIL2 −1541/ −706 segment and investigated its transcriptional regulatory properties via luciferase assays in transiently transfected HEK-293 cells (human embryonic kidney cells). The VIL2 −1541/ −706 was found to exhibit promoter activity. Furthermore, when this segment was located upstream of the VIL2 or SV40 (simian virus 40) promoters in the forward orientation, the expression levels of luciferase were dramatically enhanced. However, this transcriptional enhancement disappeared when this segment was located upstream of the promoter in the reverse orientation or downstream of the reporter gene in the forward or reverse orientation. In deletion experiments, we found several potential regulatory regions within the VIL2 −1541/ −706. When these regions were separately located upstream of the VIL2 or SV40 promoters, only the −1297/ −1186 considerably enhanced the activity of these promoters. Although the other regulatory regions exhibited significant transcriptional regulation in deletion experiments, they weakly enhanced VIL2 promoter activity and/or did not regulate SV40 promoter activity. These results suggest that the DNA sequence upstream of the VIL2 promoter functions as an enhancer in a position- and orientation-dependent manner, and the VIL2 −1297/ −1186, which acts as a key enhancer, probably regulates VIL2 transcription in combination with other potential regulatory regions located upstream of the VIL2 promoter.