Glioma　stem　cells　(GSCs)　are　thought　to　be　responsible　for　the　initiation　and　progression　of　glioblastoma　(GBM).　GBM　presents　highly　invasive　growth　with　a　very　high　recurrence　rate,　so　it　has　become　a　clinical　problem　to　be　solved　urgently.　RNAseq　demonstrates　that　thrombospondin　1　(THBS1)　acts　not　only　in　the　angiogenic　core　of　glioma　but　also　with　a　high　degree　of　invasiveness　and　infiltration.　Nevertheless,　defects　in　the　signaling　pathway　research　lead　to　a　poor　prognosis　in　glioma　patients.　To　investigate　the　relevant　molecular　mechanism　and　signal　pathway　of　glioma　stem　cell　behavior　mediated　by　THBS1,　U251　astroglioma　cells　and　GSCs　were　taken　as　model　cells　for　in　vitro　experiments.　The　biological　effects　of　THBS1　on　glioma　proliferation,　migration,　and　adhesion　were　evaluated　using　Cell　Counting　Kit-8(CCK8)　assays,　EdU　incorporation　assays,　migration　assays,　Transwell　assays,　Western　blotting,　and　RNAseq.　We　found　that　the　knockout　of　the　THBS1　gene　by　CRISPR/Cas9　promoted　proliferation　and　migration　in　U251　cells　and　GSCs,　as　well　as　influencing　cell　cycle　progression　by　regulating　the　TNF/MAPK/NF-κB　and　TGF-β/Smad　signaling　pathways.　Moreover,　U251　cells　and　GSCs　showed　different　responses　to　THBS1　knockout,　suggesting　specific　and　potential　targets　for　GSCs　in　signaling　pathways　mediated　by　THBS1.　©　2023