The invasiveness of glioma cells, a major cause of mortality in malignant brain tumors, is mediated in part by the cellular microenvironment. We have reported that in a three-dimensional matrix of type 1 collagen (3D-CL) gel, the extracellular matrix protein tenascin-C (TN) increased the invasiveness of glioma cells through the downstream production of matrix metalloproteinase (MMP)-12. In the present study, we have investigated the signaling mechanisms involved in the TN-stimulated glioma invasiveness. We found that the pan protein kinase C (PKC) inhibitor, bisindolylmaleimide I, decreased TN-enhanced glioma invasion in 3D-CL. Calphostin C, an inhibitor of conventional and novel PKC isozymes, and the relatively selective PKCdelta inhibitor rottlerin decreased TN-stimulated glioma invasiveness in a concentration- and time-dependent manner. These findings of the possible involvement of PKCdelta was supported by its translocation from the cytosol to membrane fraction in 3D-CL gel supplemented with TN as detected by western blot assays and immunofluorescence microscopy and by elevation of PKCdelta enzyme activity. Moreover, pharmacological blockade of PKCdelta decreased MMP-12 levels and glioma invasiveness. Finally, small interfering RNA to PKCdelta reduced TN-stimulated glioma invasiveness concurrent with decreased MMP-12 production. Our results implicate PKCdelta as a therapeutic target to reduce MMP-12 expression and glioma invasiveness when tumor cells are stimulated by the TN-enriched glioma microenvironment.