The identification of signaling pathways that are involved in gliomagenesis is crucial for targeted therapy design. In this study we assessed the biological and therapeutic effect of ingenol-3-dodecanoate (IngC) on glioma. IngC exhibited dose-time-dependent cytotoxic effects on large panel of glioma cell lines (adult, pediatric cancer cells, and primary cultures), as well as, effectively reduced colonies formation. Nevertheless, it was not been able to attenuate cell migration, invasion, and promote apoptotic effects when administered alone. IngC exposure promoted S-phase arrest associated with p21CIP/WAF1 overexpression and regulated a broad range of signaling effectors related to survival and cell cycle regulation. Moreover, IngC led glioma cells to autophagy by LC3B-II accumulation and exhibited increased cytotoxic sensitivity when combined to a specific autophagic inhibitor, bafilomycin A1. In comparison with temozolomide, IngC showed a mean increase of 106-fold in efficacy, with no synergistic effect when they were both combined. When compared with a known compound of the same class, namely ingenol-3-angelate (I3A, Picato®), IngC showed a mean 9.46-fold higher efficacy. Furthermore, IngC acted as a potent inhibitor of protein kinase C (PKC) activity, an emerging therapeutic target in glioma cells, showing differential actions against various PKC isotypes. These findings identify IngC as a promising lead compound for the development of new cancer therapy and they may guide the search for additional PKC inhibitors.