Temozolomide　(TMZ)　is　the　only　one　oral　first-line　chemotherapeutic　drug　for　glioblastoma　treatment.　However,　O6-methylguanine-DNA　methyltransferase　(MGMT)　can　repair　the　lethal　O6-methylguaine　(O6-MeG)　lesion　produced　by　TMZ,　thus　imparting　resistance　to　TMZ.　Currently,　the　clinical　utility　of　small　molecule　covalent　MGMT　inhibitors　is　limited　by　the　occurrence　of　severe　hematological　toxicity.　Therefore,　developing　new　strategies　for　overcoming　MGMT-mediated　resistance　is　highly　urgent.　Here,　we　explored　the　feasibility　that　modulating　Wnt/(3-catenin　signaling　pathway　in　glioblastoma　to　inhibit　MGMT　expression　to　overcome　TMZ　resistance.　From　eight　natural　products　or　approved　drugs　with　inhibitory　effects　on　Wnt/(3-catenin　pathway,　we　found　thymoquinone　(TQ)　completely　suppressed　MGMT　expression　in　glioblastoma　SF763　and　SF767　cell　lines　within　24　h.　As　expected,　TQ　exhibited　synergistic　killing　effects　with　TMZ　in　SF763　and　SF767　cells,　while　in　MGMT-negative　SF126　cells　only　additive　effect　observed.　Moreover,　TQ　remarkably　enhanced　the　inhibition　of　TMZ　on　cell　proliferation,　clone　formation,　invasion　and　migration,　and　promoted　cell　apoptosis.　In　resistant　SF763　mice　tumor　xenograft　model,　TQ　significantly　increased　the　suppression　of　TMZ　on　tumor　growth,　meanwhile　maintaining　good　biosafety.　Western　blotting　analysis　indicated　that　TQ　significantly　inhibited　the　nuclear　translocation　of　(3-catenin　and　the　expression　of　downstream　proteins　Cyclin　D1　and　MGMT.　The　addition　of　Wnt　activator　LiCl　reversed　the　nuclear　translocation　of　(3-catenin　and　the　expression　of　Cyclin　D1　and　MGMT　induced　by　TQ.　For　the　first　time,　our　findings　indicate　that　TQ　can　considerably　increase　the　sensitivity　of　glioblastoma　to　TMZ　by　interfering　Wnt/(3-catenin　pathway　to　downregulate　MGMT　expression.