ETHNOPHARMACOLOGICAL RELEVANCE: Xihuang pill, as a famous traditional Chinese medicine formula, is used for tumor treatment in China. The anti-tumor activities and mechanisms of Xihuang pill still remain unclear. AIM OF THE STUDY: The Akt/mTOR signaling pathway plays an important role in mediating cell proliferation and apoptosis in glioblastoma. This study aimed to investigate whether Xihuang pill could potentiate temozolomide-induced apoptosis of glioblastoma U87 and U251 cells in vivo and its underlying mechanisms related to Akt/mTOR pathway. MATERIALS AND METHODS: Human glioblastoma U87 and U251 xenograft models were established. Immunocytochemistry and Western blot were performed to evaluate the anti-proliferative effect, apoptosis and Akt/mTOR signaling mediators. RESULTS: The results showed that Xihuang pill (0.5, 1 g/kg) or temozolomide (10 mg/kg) treatment alone inhibited tumor growth in glioblastoma U87 and U251 xenografts. When Xihuang pill (1 g/kg) and temozolomide (10 mg/kg) were co-administrated, the activities of antitumor growth were markedly increased. Meanwhile, Xihuang pill (0.5, 1 g/kg) or temozolomide (10 mg/kg) treatment alone decreased the levels of Ki67 and PCNA expression in glioblastoma U87 and U251 xenografts. In combination treatment group, the inhibitory effects on Ki67 and PCNA expression were significantly enhanced in glioblastoma U87 and U251 xenografts compared to temozolomide treatment alone. Examining the apoptotic index by TUNEL assay showed similar results. Furthermore, Xihuang pill markedly down-regulated the Bcl-2/Bax ratio and inhibited the activation of Akt/mTOR pathway in glioblastoma U87 and U251 xenografts. In addition, no significant signs of toxicities were related to Xihuang pill and/or temozolomide treatment. CONCLUSIONS: The present study suggested that Xihuang pill might potentiate temozolomide-induced apoptosis of glioblastoma cells in vivo through inhibiting Akt/mTOR-dependent pathway.