Glioblastoma multiforme (GBM) is the most malignant primary brain tumor. High expression of vascular endothelial growth factor (VEGF) has been a hallmark of GBM. However, the regulation of VEGF expression in malignant gliomas is not well understood. We have previously reported that the transcription factor PAX6 suppresses the tumorigenecity of glioblastoma cells. In addition, there is a reverse correlation between the expression level of PAX6 and VEGF when human glioma tumors and the surrounding normal tissues were compared, suggesting that PAX6 might regulate VEGF expression. In this study, several techniques such as real-time quantitative reverse transcription PCR, enzyme-linked immunosorbent assay and immunohistochemistry were used to investigate if PAX6 regulates VEGF expression in glioma cell lines. Our results showed a significant reduction of VEGF expression in the PAX6 stable-transfected glioma cell line U251HF grown both in vitro and in subcutaneous xenografts in nude mice. We further demonstrated in two glioma cell lines (U251HF and U87) that VEGF expression was markedly suppressed by adenoviral-mediated overexpression of PAX6, as well as PTEN. Interestingly, in cells when both genes were overexpressed together, VEGF expression was further suppressed. Co-immunoprecipitation identified the existence of physical association between PAX6 and PTEN in the situation where the enhanced suppression of VEGF expression occurred. This discovery revealed a novel mechanism underlying the tumor-suppression functions of both PAX6 and PTEN in malignant gliomas. Data from this study are consistent with our previous finding in which patients with malignant gliomas have significantly more favorable outcomes when their tumors have high PAX6 and PTEN expression levels compared to patients with low expression values for either or both genes.