Mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) are activated in the majority of gliomas and contribute to tumor cell growth and survival. Sorafenib (Bay43-9006; Nexavar) is a dual-action Raf and vascular endothelial growth factor receptor inhibitor that blocks receptor phosphorylation and MAPK-mediated signaling and inhibits growth in a number of tumor types. Because our initial studies of this agent in a series of glioma cell lines showed only partial growth inhibition at clinically achievable concentrations, we questioned whether inhibition of PKC signaling using the PKC-delta inhibitor rottlerin might potentiate therapeutic efficacy. Proliferation assays, apoptosis induction studies, and Western immunoblot analysis were conducted in cells treated with sorafenib and rottlerin as single agents or in combination. Sorafenib and rottlerin reduced proliferation in all cell lines when used as single agents, and the combination produced marked potentiation of growth inhibition. Flow-cytometric measurements of cells stained with Annexin V-propidium iodide and immunocytochemical assessment of cytochrome c and apoptosis-inducing factor release demonstrated that addition of rottlerin resulted in significantly higher levels of apoptosis than sorafenib alone. In addition, the combination of sorafenib and rottlerin reduced or completely inhibited the phosphorylation of extracellular signal-regulated kinase and Akt and down-regulated cell cycle regulatory proteins such as cyclin-D1, cyclin-D3, cyclin-dependent kinase (cdk)4, and cdk6 in a dose- and time-dependent manner. Our results clearly indicate that inhibition of PKC-delta signaling enhances the antiproliferative effect of sorafenib in malignant human glioma cell lines and support the examination of combinations of signaling inhibitors in these tumors.