We reported that PAX6 suppresses glioblastoma cell growth in vivo and anchorage-independent growth without significant alteration of cell proliferation in vitro, suggesting that PAX6 may alter the tumor microenvironment. Because we found that PAX6 downregulates expression of the gene encoding vascular endothelial growth factor A (VEGFA) in glioma cells, we used a subcutaneous xenograft model to verify PAX6 suppression of VEGFA-induced angiogenesis based on CD31-immunostaining of endothelial cells. The results showed a significant reduction of VEGFA at the transcription level in PAX6-transfected cells in xenografts and PAX6 has a suppressive effect on the microvascular amplification typically seen in glioblastoma. We showed that PAX6 suppression of VEGFA expression requires its DNA binding-domain. The C-terminal truncation mutant of PAX6, however, did not show the dominant negative function in regulating VEGFA expression that it showed previously in regulating MMP2 expression. In the glioma cell line U251HF, we further determined that blocking the PI3K/Akt signaling pathway with either adenoviral-mediated PTEN expression or LY294002 enhanced PAX6-mediated suppression of VEGFA in an additive manner; thus, PAX6-mediated suppression of VEGFA is not via the canonical pathway through HIF1A. These two VEGFA-regulatory pathways can also be similarly modulated in another malignant glioma cell line, U87, but not in LN229 where the basal VEGFA level is low and PTEN is wild-type. PAX6 suppression of VEGFA appears to be blocked in LN229. In conclusion, our data showed that PAX6 can initiate in glioma cells a new signaling pathway independent of PI3K/Akt-HIF1A signaling to suppress VEGFA expression.