Exploring novel therapeutic approaches is critically needed for the treatment of glioblastoma, due to concerns over its invasiveness and drug resistance. Trivalent arsenic derivative (arsenite, As^III), and two bufadienolide compounds, arenobufagin (Areno) and gamabufotalin (Gama) have been reported to induce cytotoxicity in glioblastoma cells. Herein, the cytotoxicity of As^III combined with Areno or Gama was evaluated in the human glioblastoma cell line U-87. A dose-dependent cytotoxicity was observed in the cells treated by As^III, Areno and Gama, respectively. Enhancement of cytotoxicity was induced by As^III combined with Areno or Gama, and synergistic cytotoxic effects of clinically achieved concentrations of As^III combined with Areno were further observed. Apoptosis induction accompanied by a downregulation of proform of caspase-9 and caspase-3 was observed following the treatment with the combined regimen of As^III and Areno. The combined regimen also caused enhanced necrosis as evidenced by a clear increase in LDH release and propidium iodide-positive cell populations. In comparison to each single drug treatment, the combined regimen apparently downregulated the expression level of p-Akt, p-mTOR; and upregulated the expression level of LC3. Collectively, the combined regimen of As^III and Areno exhibited a unique multivalent cytocidal effects against U-87 cells by triggering apoptotic, necrotic and autophagic cell death, suggesting that developing a new combination regimen of As^III and Areno may offer benefits to patients with glioblastoma.