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.