Hypericum perforatum is a medicinal herb possessing ability for protecting neurons from oxidative stress. Since nitric oxide (NO) may be protective against oxidative stress-induced cell death as occurs in glucose deprivation (GD)-induced neurotoxicity, whether a standardized extract of H. perforatum (HP) increases the NO-mediated neuroprotective effect in GD-PC12 cells was investigated. Induced death in PC12 cells by GD exposure for 18 h was partially prevented by cell incubation with sodium nitroprusside (SNP), a NO-donor. SNP increased survival and nitrite production in GD-cells in a concentration-dependent manner. Co-incubation of cells with 10 μM SNP plus 50-100 μg/ml HP under GD insult significantly prevented GD-induced cell death to a higher extent than SNP alone as shown by an augmentation of cell survival and intracellular bcl-2 levels and a decrease of lipid peroxidation, caspase-3 activation and PARP cleavage. Cytoprotection by the NO-donor was almost abolished by the use of a NO scavenger and potentiated by the presence of superoxide dismutase. SNP and/or HP neuroprotection on GD-cells was significantly reversed by rotenone treatment. These results suggest that: (1) SNP could protect PC12 cells from GD-induced cytotoxicity through NO generation and (2) the enhancement of the SNP-mediated neuroprotective effect on GD-cells by HP might arise in part through scavenging of reactive oxygen species (ROS) and inhibition of mitochondrial dysfunction associated with the hypoglycemic episode. This current finding might highlight the development of therapeutic strategies aimed at manipulating NO-donors in combination with herb supplements containing ROS scavenger compounds for prophylaxis from brain ischemia.