The actions of insulin-like growth factors (IGFs) are regulated at various levels. One mechanism involves binding to IGF Binding Protein-3 (IGFBP-3) for transport, thus governing bioavailability. IGFBP3 transcription is modulated by many hormones and agents that stimulate or inhibit growth. We have previously shown in pediatric and adult cohorts, a correlation between IGFBP-3 serum levels and two single nucleotide polymorphisms (SNPs) located within the minimal promoter (-202 A/C and -185 C/T). Functionality of these SNPs was further explored in hepatic adenocarcinoma-derived SK-HEP-1 cells using transient transfections of luciferase constructs driven by different haplotypes of the IGFBP3 promoter. Basal luciferase activity revealed a significant haplotype-dependent transcriptional activity (at nucleotides -202 and -185: AC>CC, p<0.001; AC>CT, p<0.001; AC>AT, p< 0.001). Insulin treatment produced a similar haplotype-dependence of luciferase activity (AC>CC, p=0.002; AC>CT, p<0.001; AC>AT, p=0.011). However induction ratios (insulin/control) for CC and AT were significantly higher compared to AC and CT (CC>AC, p=0.03; CC>CT, p=0.03; AT>AC, p=0.03; AT>CT, p=0.04). Gel retardation assays were used to identify Upstream Stimulatory Factor-1 and -2 (USF-1, USF-2), methylation-dependent binding to E-box motifs located between the SNPs. Mutation of the USF binding site resulted in a significant loss of insulin stimulation of luciferase activity in the transfection assay. Chromatin immunoprecipitation with anti-USF-1/-2 showed an enrichment of IGFBP3 promoter in insulin-treated cells compared to unstimulated cells. Bisulfite sequencing of genomic DNA revealed that CpG methylation in the region of USF binding was haplotype-dependent. In summary, we report a methylation-dependent USF binding site influencing the basal and insulin-stimulated transcriptional activity of the IGFBP3 promoter.