JSI-124 (cucurbitacin I) is a selective inhibitor of Janus kinase/signal transducer and activator of transcription 3(JAK/STAT3) and has been shown to exert anti-proliferative and anti-tumor properties both in vitro and in vivo. As STAT3 activation has been implicated in the development of glioma, we investigated the therapeutic efficacy of JSI-124 on glioblastoma multiforme (GBM) by interfering with STAT3 pathway. In present study, two GBM cell lines, U251 and A172 cells, were treated with JSI-124. The results showed that the cell growth was inhibited significantly in a dose-and time-dependent manner. Further investigation illustrated that the levels of phosphorylated-STAT3 were decreased in GBM cells treated by JSI-124, concomitant with apoptosis augment and cell cycle arrest. Specially, JSI-124 induced G(2)/M accumulation via downregulation of cyclin B1 and cdc2 expression. Together these results suggested that inhibition of STAT3 by JSI-124 is a potential strategy for the development of the new glioblastoma multiforme therapeutics.