Vascular endothelial growth factor (VEGF) is a key angiogenic molecule that plays an important role in the growth and metastasis of many types of human cancer, including pancreatic adenocarcinoma. In this study, we explored the regulation of VEGF in human pancreatic cancer cells. Over 70% of the human pancreatic cancer cell lines studied in vitro secreted constitutively high levels of VEGF. High VEGF-secreting cells also generally expressed an elevated steady-state level of VEGF mRNA. Kinetic analysis revealed that the elevated steady-state level of VEGF mRNA was due to enhanced VEGF gene transcription and increased constitutive VEGF promoter activity. Deletive mutation analyses of the VEGF promoter revealed that the region from -109 to -38 bp was essential for constitutive VEGF promoter activity. Further deletion and point mutation analyses indicated that mutation of individual or all of the putative Sp1 binding sites reduced or eliminated the constitutive VEGF promoter activity and abrogated the differential activity of the promoter in high and low VEGF-expressing cells. Consistent with the constitutive VEGF transcription activation, a high level of constitutive Sp1 expression and activity was detected in pancreatic cancer cell lines and pancreatic cancer tissue specimens overexpressing VEGF. Collectively, our data demonstrated that constitutive Sp1 activation is essential for the differential overexpression of VEGF, which in turn plays an important role in the angiogenesis and progression of human pancreatic cancer.