Malignant gliomas are the most common primary brain tumor and have a poor clinical prognosis. 1, 3-Bis (2-chloroethyl)-1-nitrosourea (BCNU) is an alkylating agent that is commonly used in glioma therapy. However, BCNU chemotherapy often fails due to drug resistance. To gain better understanding of molecular mechanisms underlying the drug resistance of glioma, a BCNU-resistant variant (C6R) of C6 rat glioma cells was selected and characterized. The established C6R cells were resistant to BCNU-induced cell death and cell cycle arrest as confirmed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide reduction assay and flow cytometric analysis of DNA content. C6R cells showed an increased expression of common drug resistance-related genes such as O6-methylguanine-DNA methyltransferase and multiple drug resistance 1. In contrast, C6R cells showed a decreased expression of glial fibrillary acidic protein, therefore, displaying shorter cellular processes compared with parental C6 cells. More importantly, in conjunction with the morphological changes, the expression of lipocalin-2 (lcn2), a 25-kDa secreted proapoptotic protein, was markedly reduced in the BCNU-resistant C6R cells. However, there was no significant change in the expression of lcn2 receptors. Addition of recombinant LCN2 protein or introduction of lcn2 cDNA significantly increased the sensitivity of C6 cells and human glioma cells to BCNU or other anticancer drugs, while knockdown of lcn2 expression by antisense cDNA transfection decreased the sensitivity. When lcn2 was re-expressed in C6R cells, the BCNU sensitivity was restored. Lcn2 enhanced BCNU-induced Akt dephosphorylation providing a molecular basis of apoptosis sensitization. These results suggest that LCN2 protein may be involved in glioma drug resistance and may provide a new approach to sensitizing glioblastoma to chemotherapy.