Background: Na/K-ATPase (NKA) is inhibited by perillyl alcohol (POH), a monoterpene used in the treatment of tumors, including brain tumors. The NKA α1 subunit is known to be superexpressed in glioblastoma cells (GBM). This isoform is embedded in caveolar structures and is probably responsible for the signaling properties of NKA during apoptosis. In this work, we showed that POH acts in signaling cascades associated with NKA that control cell proliferation and/or cellular death. Methods: NKA activity was measured by the amount of non-radioactive Rb+ incorporation into cultured GBM cell lines (U87 and U251) and non-tumor cells (mouse astrocytes and VERO cells). Cell viability was measured by lactate dehydrogenase levels in the supernatants of POH-treated cells. Activated c-Jun N-terminal Kinase (JNK) and p38 were assessed by western blotting. Apoptosis was detected by flow cytometry and immunocytochemistry, and the release of interleukins was measured by ELISA. Results: All four cell types tested showed a similar sensitivity for POH. Perillic acid (PA), the main metabolite of POH, did not show any effect on these cells. Though the cell viability decreased in a dose-dependent manner when cells were treated with POH, the maximum cytotoxic effect of PA obtained was 30% at 4 mM. 1.5 mM POH activated p38 in U87 cells and JNK in both U87 and U251 cells as well as mouse astrocytes. Dasatinib (an inhibitor of the Src kinase family) and methyl β-cyclodextrin (which promotes cholesterol depletion in cell membranes) reduced the POH-induced activation of JNK1/2 in U87 cells, indicating that the NKA-Src complex participates in this mechanism. Inhibition of JNK1/2 by the JNK inhibitor V reduced the apoptosis of GBM cells that resulted from POH administration, indicating the involvement of JNK1/2 in programmed cell death. 1.5 mM POH increased the production of interleukin IL-8 in the U251 cell supernatant, which may indicate a possible strategy by which cells avoid the cytotoxic effects of POH. Conclusions: A signaling mechanism mediated by NKA may have an important role in the anti-tumor action of POH in GBM cells.