We have investigated the effectiveness of higenamine in the treatment of malignant glioma, and explored its possible mechanism in C6 glioma cells. The efficacy of higenamine on viability of cells, apoptosis, cell cycle arrest, DNA fragmentation, and biochemical markers was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, enzyme-linked immunosorbent assay, and Western blotting. The biochemical markers investigated included the effect of higenamine on the expression of phosphoinositide-3-kinase/protein kinase B, B-cell lymphoma 2, BCL2- associated X protein, cysteine-aspartic proteases-3 and -9 proteins. The translocation of nuclear factor-kappa B from the nucleus was also analyzed. Results revealed that higenamine induced cytotoxic and antiproliferative effects on the C6 glioma cells. Higenamine led to cell arrest at G2/M phase of cell cycle and lowered cell count at S-phase. The maximum extent of DNA fragmentation was observed after 72 h exposure of higenamine. Nuclear translocation of nuclear factorkappa B was attenuated after higenamine treatment in the C6 glioma cells. The results also revealed that higenamine significantly modulated the phosphoinositide-3-kinase/protein kinase B signaling cascade. Also, higenamine elevated the cysteine-aspartic proteases-3 and -9 and BCL2-associated X protein, and downregulated B-cell lymphoma 2 expression in the C6 glioma cells. Overall, the investigation suggests higenamine modulation of phosphoinositide-3-kinase/protein kinase B signaling pathway, nuclear factor-kappa B nuclear translocation, and caspase cascade in the C6 glioma cells.