Y-box binding protein 1 (YB-1) is manifested as its involvement in cell proliferation and differentiation and malignant cell transformation.Overexpression of YB-1 is associated with glioma progression and patient survival.The aim of this study is to investigate the influence of YB-1 knockdown on glioma cell progression and reveal the mechanisms of YB-1 knockdown on glioma cell growth,migration,and apoptosis.It was found that the knockdown of YB-1 decreased the mRNA and protein levels of YB-1 in U251 glioma cells.The knockdown of YB-1 significantly inhibited cell proliferation,colony formation,and migration in vitro and tumor growth in vivo.Proteome and phosphoproteome data revealed that YB-1 is involved in glioma progression through regulating the expression and phosphorylation of major proteins involved in cell cycle,adhesion,and apoptosis.The main regulated proteins included CCNB1,CCNDBP1,CDK2,CDK3,ADGRG1,CDH-2,MMP14,AIFM1,HO-1,and BAX.Furthermore,it was also found that YB-1 knockdown is associated with the hypo-phosphorylation of ErbB,mTOR,HIF-1,cGMP-PKG,and insulin signaling pathways,and proteoglycans in cancer.Our findings indicated that YB-1 plays a key role in glioma progression in multiple ways,including regulating the expression and phosphorylation of major proteins associated with cell cycle,adhesion,and apoptosis.