Berberine has a wide range of biochemical and pharmacologic effects, including antitumor activity, but the mechanisms involved in berberine-induced apoptosis remain unclear. The purpose of the present study was to investigate the changes in oxidative stress and endoplasmic reticulum (ER)-related molecules, which are closely associated with cell death-signaling transduction pathways, in human glioblastoma T98G cells treated with berberine. Berberine significantly decreased the cell viability of T98G cells in a dose-dependent manner. Berberine increased the production of reactive oxygen species (ROS) and level of intracellular Ca²⁺. Berberine induced ER stress as evidenced by the detection of ER stress-associated molecules such as phosphorylated protein kinase-like ER kinase, eukaryotic translation initiation factor-2α, glucose-regulated protein 78/immunoglobulin heavy chain-binding protein, and CCAAT/enhancer-binding protein (C/EBP)-homologous protein/growth arrest and DNA damage-inducible gene 153, which was associated with the activation of caspase-3. Furthermore, the administration of the antioxidants, N-acetylcysteine and glutathione, reversed berberine-induced apoptosis. Berberine also markedly enhanced apoptosis in T98G cells through the induction of a higher ratio of Bax/Bcl-2 proteins, disruption of the mitochondrial membrane potential, activation of caspase-9 and -3, and cleavage of the poly(ADP-ribose) polymerase (PARP). The inhibition of ER stress using salubrinal led to an increased the level of Bcl-2, whereas the level of Bax, cleavage of procaspase-9 and -3, and PARP were decreased when compared with cells treated with berberine alone, indicating that berberine-induced apoptosis is associated with mitochondrial dysfunction. These results demonstrate that berberine induces apoptosis via ER stress through the elevation of ROS and mitochondrial-dependent pathway in human glioblastoma T98G cells.