Chloroethylnitrosoureas　(CENUs)　are　bifunctional　antitumor　alkylating　agents,　which　exert　their　antitumor　activity　through　inducing　the　formation　of　dG-dC　interstrand　crosslinks　(ICLs)　within　DNA　double　strand.　However,　the　complex　process　of　tumor　biology　enables　tumor　cells　to　escape　the　killing　triggered　by　CENUs,　as　for　instance　with　the　detoxifying　activity　of　O-6-methylguanine　DNA　methyltransferase　(MGMT)　to　accomplish　DNA　damage　repair.　Considering　the　fact　that　most　tumor　cells　highly　depend　on　aerobic　glycolysis　to　provide　energy　for　survival　even　in　the　presence　of　oxygen　(Warburg　effect),　inhibition　of　aerobic　glycolysis　may　be　an　attractive　strategy　to　overcome　the　resistance　and　improve　the　chemotherapeutic　effects　of　CENUs.　Especially,　3-bromopyruvate　(3-BrPA),　a　small　molecule　alkylating　agent,　has　been　emerged　as　an　effective　glycolytic　inhibitor　(energy　blocker)　in　cancer　treatment.　In　view　of　its　tumor　specificity　and　inhibition　on　cellular　multiple　targets,　it　is　likely　to　reduce　the　chemoresistance　when　chemotherapeutic　drugs　are　combined　with　3-BrPA.　In　this　study,　we　investigated　the　effects　of　3-BrPA　on　the　chemosensitivity　of　two　human　hepatocellular　carcinoma　(HCC)　cell　lines　to　the　cytotoxic　effects　of　1,3-bis(2-chloroethyl)1-nitrosourea　(BCNU)　and　the　underlying　molecular　mechanism.　The　sensitivity　of　SMMG7721　and　HepG2　cells　to　BCNU　was　significantly　increased　by　2　h　pretreatment　with　micromolar　dosage　of　3-BrPA.　Moreover,　3-BrPA　decreased　the　cellular　ATP　and　GSH　levels,　and　extracellular　lactate　excreted　by　tumor　cells,　and　the　effects　were　more　effective　when　3-BrPA　was　combined　with　BCNU.　Cellular　hexokinase-II　(HK-II)　activity　was　also　reduced　after　exposure　to　the　treatment　of　3-BrPA　plus　BCNU.　Based　on　the　above　results,　the　effects　of　3-BrPA　on　the　formation　of　dG-dC　ICLs　induced　by　BCNU　was　investigated　by　stable　isotope　dilution　high-performance　liquid　chromatography　electrospray　ionization　tandem　mass　spectrometry　(HPLC-ESI-MS/MS).　The　results　indicated　that　BCNU　produced　higher　levels　of　dG-dC　ICLs　in　SMMC-7721　and　HepG2　cells　pretreated　with　3-BrPA　compared　to　that　without　3-BrPA　pretreatment.　Notably,　in　MGMT-deficient　HepG2　cells,　the　levels　of　dG-dC　ICLs　were　significantly　higher　than　MGMT-proficient　SMMC-7721　cells.　In　general,　these　findings　revealed　that　3-BrPA,　as　an　effective　glycolytic　inhibitor,　may　be　considered　as　a　potential　clinical　chemosensitizer　to　optimize　the　therapeutic　index　of　CENUs.