An increase in MMP-9 gene expression and enzyme activity with stimulating the migration of GBM8401 glioma cells via wound healing assay by 12-O-tetradecanoylphorbol-13-acetate (TPA) was detected in glioblastoma cells GBM8401. TPA-induced translocation of protein kinase C (PKC)α from the cytosol to membranes, and migration of GBM8401 elicited by TPA was suppressed by adding the PKCα inhibitors, GF109203X and H7. Activation of extracellular signal-regulated kinase (ERK) and c-Jun-N-terminal kinase (JNK) by TPA was identified, and TPA-induced migration and MMP-9 activity was significantly blocked by ERK inhibitor PD98059 and U0126, but not JNK inhibitor SP600125. Activation of NF-κB protein p65 nuclear translocation and IκBα protein phosphorylation with increased NF-κB-directed luciferase activity by TPA were observed, and these were blocked by the PD98059 and IkB inhibitor BAY117082 accompanied by reducing migration and MMP-9 activity induced by TPA in GBM8401 cells. Transfection of GBM8401 cells with PKCα siRNA specifically reduced PKCα protein expression with blocking TPA-induced MMP-9 activation and migration. Additionally, suppression of TPA-induced PKCα/ERK/NK-κB activation, migration, and MMP-9 activation by flavonoids including kaempferol (Kae; 3,5,7,4′-tetrahydroxyflavone), luteolin (Lut; 5,7,3′4′-tetrahydroxyflavone), and wogonin (Wog; 5,7-dihydroxy-8-methoxyflavone) was demonstrated, and structure–activity relationship (SAR) studies showed that hydroxyl (OH) groups at C4′ and C8 are critical for flavonoids' action against MMP-9 enzyme activation and migration/invasion of glioblastoma cells elicited by TPA. Application of flavonoids to prevent the migration/invasion of glioblastoma cells through blocking PKCα/ERK/NF-κB activation is first demonstrated herein. J. Cell. Physiol. 225: 472–481, 2010. © 2010 Wiley-Liss, Inc.