DNA　interstrand　cross-links　(ICLs)　are　essential　for　the　antitumor　activity　of　chloroethylnitrosoureas　(CENUs).　Commonly,　CENUs　resistance　is　mainly　considered　to　be　associated　with　O-6-methylguanine-DNA　methyltransferase　(MGMT)　within　tumors.　Bypassing　the　MGMT-mediated　resistance,　to　our　knowledge,　herein,　we　first　utilized　a　novel　glycolytic　inhibitor,　3-bromopyruvate　(3-BrPA),　to　increase　the　cytotoxic　effects　of　1,3-bis(2-chloroethyl)-1-nitrosourea　(BCNU)　to　human　glioma　cells　based　on　the　hypothesis　that　blocking　energy　metabolism　renders　tumor　cells　more　sensitive　to　chemotherapy.　We　found　3-BrPA　significantly　increased　the　cell　killing　by　BCNU　in　human　glioma　SF763　and　SF126　cell　lines.　Significantly　decreased　levels　of　extracellular　lactate,　cellular　ATP　and　glutathione　(GSH)　were　observed　after　3-BrPA　treatment,　and　the　effects　were　more　remarkable　with　3-BrPA　in　combination　with　BCNU.　Considering　that　the　role　of　ATP　and　GSH　in　drug　efflux,　DNA　damage　repair　and　drug　inactivation,　we　determined　the　effect　of　3-BrPA　on　the　formation　of　dG-dC　ICLs　induced　by　BCNU　using　stable　isotope　dilution　high-performance　liquid　chromatography　electrospray　ionization　tandem　mass　spectrometry　(HPLC-ESI-MS/MS).　As　expected,　the　levels　of　lethal　dG-dC　ICLs　induced　by　BCNU　were　obviously　enhanced　after　3-BrPA　pretreatment.　Based　on　these　results,　3-BrPA　and　related　glycolytic　inhibitors　may　be　promising　to　enhance　the　cell　killing　effect　and　reverse　the　clinical　chemoresistance　of　CENUs　and　related　antitumor　agents.