Tumor　resistance　seriously　hinders　the　clinical　application　of　chloroethylnitrosoureas　(CENUs),　such　as　O6-methylguanine-DNA　methylguanine　(MGMT),　which　can　repair　O6-alkyl　lesions,　thereby　inhibiting　the　formation　of　cytotoxic　DNA　interstrand　cross-links　(ICLs).　Metabolic　differences　between　tumor　and　normal　cells　provide　a　biochemical　basis　for　novel　therapeutic　strategies　aimed　at　selectively　inhibiting　tumor　energy　metabolism.　In　this　study,　the　energy　blocker　lonidamine　(LND)　was　selected　as　a　chemo-sensitizer　of　nimustine　(ACNU)　to　explore　its　potential　effects　and　underlying　mechanisms　in　human　glioblastoma　in　vitro　and　in　vivo.　A　series　of　cell-level　studies　showed　that　LND　significantly　increased　the　cytotoxic　effects　of　ACNU　on　glioblastoma　cells.　Furthermore,　LND　plus　ACNU　enhanced　the　energy　deficiency　by　inhibiting　glycolysis　and　mitochondrial　function.　Notably,　LND　almost　completely　downregulated　MGMT　expression　by　inducing　intracellular　acidification.　The　number　of　lethal　DNA　ICLs　produced　by　ACNU　increased　after　the　LND　pretreatment.　The　combination　of　LND　and　ACNU　aggravated　cellular　oxidative　stress.　In　resistant　SF763　mouse　tumor　xenografts,　LND　plus　ACNU　significantly　inhibited　tumor　growth　with　fewer　side　effects　than　ACNU　alone.　Finally,　we　proposed　a　new　“HMAGOMR”　chemo-sensitizing　mechanism　through　which　LND　may　act　as　a　potential　chemo-sensitizer　to　reverse　ACNU　resistance　in　glioblastoma:　moderate　inhibition　of　hexokinase　(HK)　activity　(H);　mitochondrial　dysfunction　(M);　suppressing　adenosine　triphosphate　(ATP)-dependent　drug　efflux　(A);　changing　redox　homeostasis　to　inhibit　GSH-mediated　drug　inactivation　(G)　and　increasing　intracellular　oxidative　stress　(O);　downregulating　MGMT　expression　through　intracellular　acidification　(M);　and　partial　inhibition　of　energy-dependent　DNA　repair　(R).　©　2024　American　Chemical　Society.