The human SLC2A10 gene encodes the high-affinity glucose transporter 10 (GLUT10) and is widely expressed in adult tissues, including organs which play major roles in glucose homeostasis. Its function and genomic location in a region linked to Type 2 diabetes susceptibility are consistent with a potential role in Type 2 diabetes. Analysis of the CpG-rich promoter revealed the presence of two major transcription start points with differential use in tissues and cell lines. Mapping of transcriptionally active regions in the 5' flanking sequence identified a region, located between nucleotides -70 and -14 (relative to the major transcription start point) as the SLC2A10 basal promoter. This sequence harbors consensus binding sites for Sp, AP2alpha, and other transcription factors. A juxtaposed Sp/AP2alpha motif located between -25 and -11 is critical for core promoter function. In cells expressing Sp and AP2 factors, the two motifs are required for maximal activation of the basal promoter. In cells lacking AP2alpha, transcription is dependent on the integrity of the Sp site. Using electrophoresis mobility shift assays, we demonstrate that Sp1 and Sp3 bind to the GC-box in site 5 forming specific complexes. In addition, a silencer region is present upstream of -696 which down-regulates SLC2A10 promoter activity independently of its distance to the transcript start site.