Chloroethylnitrosoureas　(CENUs)　exert　antitumor　activity　via　producing　dG-dC　interstrand　crosslinks　(ICLs).　However,　tumor　resistance　make　it　necessary　to　find　novel　strategies　to　improve　the　therapeutic　effect　of　CENUs.　2-Deoxy-D-glucose　(2-DG)　is　a　well-known　glycolytic　inhibitor,　which　can　reprogram　tumor　energy　metabolism　closely　related　to　tumor　resistance.　Here,　we　investigated　the　chemosensitization　effect　of　2-DG　on　l,3-bis(2-chloroethyl)-1-nitrosourea　(BCNU)　against　glioblastoma　cells　and　the　underlying　mechanisms.　We　found　that　2-DG　significantly　increased　the　inhibitory　effects　of　BCNU　on　tumor　cells　compared　with　BCNU　alone,　while　2　-DG　showed　no　obvious　enhancing　effect　on　the　BCNU-induced　cytotoxicity　for　normal　HaCaT　and　HA1800　cells.　Proliferation,　migration　and　invasion　determinations　presented　the　same　trend　as　survival　on　tumor　cells.　2-DG　plus　BCNU　increased　the　energy　deficiency　through　a　more　effective　inhibition　of　glycolytic　pathway.　Notably,　the　combination　of　2-DG　and　BCNU　aggravated　oxidative　stress　in　glioblastoma　cells,　along　with　a　significant　decrease　in　glutathione　(GSH)　levels,　and　an　increase　in　intracellular　reactive　oxygen　species　(ROS).　Subse-quently,　we　demonstrated　that　the　combination　treatment　led　to　increased　apoptosis　via　activating　mitochondria　and　endoplasmic　reticulum　stress　(ERS)　related　apoptosis　pathways.　Finally,　we　found　that　the　dG-dC　level　was　significantly　increased　after　2-DG　pretreatment　compared　to　BCNU　alone　by　HPLC-ESI-MS/MS　analysis.　Finally,　in　vivo,　2-DG　plus　BCNU　significantly　suppressed　tumor　growth　with　lower　side　effects　compared　with　BCNU　alone　in　tumor-bearing　mice.　In　summary,　we　proposed　that　2-DG　may　have　potential　to　increase　the　sensitivity　of　glioblastoma　cells　to　BCNU　by　regulating　glycolysis,　ROS　and　ERS　pathways　in　clinical　setting.