Abstract: Objective: To confirm that BCNU resistance is induced by hypoxic preconditioning and to clarify the role of neuronal nitric oxide synthase (nNOS) in hypoxic preconditioning. Methods: SK-N-SH cells were pretreated using 300 μMCoCl₂ for 3 h, then 24 hours later the cells were treated with 30ug/ml BCNU. MTT assay was used to detect cell proliferation and FACS was used to determine the apoptotic index. RT-PCR was used to detect expression of nNOS and other apoptosis-related genes after hypoxic preconditioning. Results: Pretreatment with 300 μM CoCl₂ for 3 h followed by treatment with BCNU 24 hours later significantly increased proliferation and decreased the apoptotic index compared to BCNU treatment without hypoxic pretreatment. Expression of nNOS was upregulated after preconditioning, while expression of the Bcl-2 and Apaf-1 genes was upregulated and downregulated, respectively. These changes can be arrested by 7-NI, an inhibitor of nNOS. Conclusion: The 3-h hypoxic pretreatment can induce BCNU resistance in glioma cells in vitro. During the preconditioning, nNOS can regulate the expression of the Bcl-2 and Apaf-1 gene, thus playing an important role.