Abstract BACKGROUND Hippo/YAP signaling pathway has emerged as an important driver of GBM. However, the clinical significance and expression correlation of YAP in GBM is still unknown. The purpose of this study is to elucidate the regulatory functions of YAP in response to tumorspheres (TS) in GBM. Material and METHODS In GBM-tumorspheres (TS), mRNA levels of Yap1 and TAZ were determined by RNA-Seq, and nuclear YAP1 expression level and its correlation with tumor aggressiveness were assessed through nucleus cytosolic fractionation, quantitative confocal microscopy, western blot, and TEAD4 reporter assay. Cell proliferation, stemness, and invasive properties were also measured using YAP siRNA and its inhibitor Verteporfin to reveal the effect of YAP on glioma progression. In orthotopic xenograft models, the inhibition of YAP function were tested. RESULTS All GBM-TS exhibited various phosphorylation states of YAP but no significant differences regarding the expression of YAP. Among them, TS15-88 cell line showed relatively low YAP phosphorylation in the quantified immunoblot results. Indeed, TS with reduced YAP phosphorylation was associated with aggressive cancer phenotypes. Predominant nuclear localization of YAP and binding of YAP/TAZ to TEAD in the nucleus was evident in the TS15-88 cells. The YAP knockdown (KD) by siRNA significantly resulted decrease in TS proliferation through WST and ATP assay and values were validated again through YAP inhibitor, Verteporfin (VP). YAP KD attenuated not only the invasion and stemness of TS but also caused a reduction in tumor volume and prolonged the overall survival of mice. CONCLUSION We showed alteration of gene activities by YAP and nuclear localization of YAP/TAZ complex contributes to GBM progression. Moreover, thus tuning of Yap activity results a marked suppression in aggressive phenotypes of cancer. These associations emphasize that the YAP signaling network highly suggests new therapeutic opportunities in diagnosing and treating GBM.