Glioblastoma (GBM) is the most aggressive cerebral gliomas. Moreover, the overall prognosis of GBM is still little. Baicalein (BA) is a flavonoid derived from the Scutellaria baicalensis root, and has historically been used in anticancer therapies. However, its apoptosis role and related mechanisms in GBM has not yet been researched clearly. Thus, this study aimed to investigate the effects of BA on human GBM U251 cell line. The effects of BA on proliferation of U251 cells were measured by Cell Counting Kit-8 assay. Cellular apoptosis was detected by flow cytometry with annexin V-FITC/propidium iodide staining. The expression of apoptosis-related protein Bcl-2, Bax and cleaved-caspase3 was detected by quantitative real-time PCR and western blot. The expression of nuclear p65 protein, the active subunit of nuclear factor-kappa B (NF-B), was determined by immunofluorescence and western blot. Our results showed that the viability of U251 cells significantly decreased in a time- and dose-dependent manner after treated with BA, and the apoptotic ratio of BA-treated groups was significantly higher than that of control groups. Furthermore, the expression of NF-kB-p65 in the nucleus was remarkably reduced, and the activity of NF-kB-p65 was remarkably inhibited after BA treatment. Combined treatment with a NF-kB-P65 inhibitor (QNZ) and BA resulted in the synergistic reduction of Bcl-2 expression and then increase of Bax and cleaved-caspase3 expression; and the viability of U251 cells was also inhibited. In conclusion, BA inhibits GBM cells viability and induces apoptosis via inhibit the activity of NF-kB-p65, suggesting that BA is a potential therapeutic agent for GBM.