Objective：This study aims to construct the lentiviral vector carrying the cDNA encoding neuronal nitric oxide synthase（nNOS）of rat and to detect its expression and catalytic function. Methods：The cDNA of nNOS was extracted by RT-PCR followed by eukaryotic expression vector of CDH-GFP for cloning．After confirming the cDNA of nNOS by sequencing，cDNA of nNOS was cloned into the lentiviral vector pCDH-GFP for constructing recombinant vector pCDH-GFP/nNOS．The plasmid pCDH-GFP/nNOS was then transfected into 293T cells in the presence ofhelper plasmids by Lipofectamine 2000 mediation for packing lentiviral particles LV-nNOS-GFP．The cultured neuronal stem cells（NSCs）were used to verify whether LV-nNOS-GFP can infect NSCs by co- incubation with LV-nNOS-GFP Afterwards，LV-nNOS-GFP was injected into the dentate gyrys（DG）of the hippocampus to measure the expression of nNOS and the generation of nitric oxide（NO）. Results：The full length cDNA of nNOS could besuccessfully amplified and constructed into the recombinant lentiviral vector pCDH-GFP/nNOS. The packaged LV-nNOS-GFP successfully infected cultured NSCs and neurons in the hippocampal DG. The expressed nNOS was catalytic effective in catalyzing NO generation. Conclusion：The lentiviral vector of LV-nNOS-GFP was constructed successfully with functional nNOSexpression after infection.