Objective To explore whether or not the glioma U251 stem cell regulates its resistance to TMZ by the FOXO3a/β-catenin pathway.Methods The U251 stem cells were divided into the TMZ group(100 μmol/IL TMZ treated cells)and the control group.The Western blot and real-time quantitative reverse transcription-PCR(qRT-PCR)were used to detect the expression levels of FOXO3a,β-catenin,Nestin,CD133 and Sox2 under the TMZ action in the two groups.The stem cell pelletization experiment was used to verify the resistance of U251 stem cell on TMZ.Recombinant viral vectors pHY-FOXO3a,pHY-β-catenin-KD and PHy-β-Catenin-KD were constructed by embedding FOXO3a/β-catenin interference sequences into lentivirus pHY-LV-KD1.1 expression vector and transfected into U251 stem cells.The change of stem cell clone pellets number was measured under TMZ action.The effects of knockdown FOXO3a and β-catenin on the characteris-tics and drug resistance of U251 stem cells were observed.Results The Western blot and qRT-PCR results showed that compared with the control group,the expression levels of FOXO3a,β-catenin,Nestin,CD133 and Sox2 protein in the TMZ group were increased,the Nestin,CD133,Sox2 mRNA expression levels were in-creased(P＜0.05).The clone formation experiment results showed that the majority of survival cells on 5 d after TMZ treatment were the stem cells,indicating that the U251 stem cells could better tolerate TMZ.Knoc-king down FOXO3a and β-catenin could reduce the U251 stem cell populations number,indicating that its re-sistance to TMZ was weakened.Conclusion The FOXO3a/β-catenin pathway could regulates the characteris-tics of U251 stem cell and TMZ resistance.