The present study aimed to investigate the role of nitric oxide (NO) against perinatal hypoxic-ischemic brain damage (HIBD) in rats by electroacupuncture (EA) and to examine its potential neuroprotective mechanism. NO content, the number of positive cells, neuronal nitric oxide synthase (nNOS) and nuclear factor-κB (NF-κB) in rat cortex cells were determined. The results demonstrated that treatment with EA significantly downregulated the NO content in the cortex cells (*P<0.05, **P<0.01, compared with the control groups) and alleviated cell damage in the cortex of rats with HIBD. The activator, S-adenosyl-L-methionine and the inhibitor, hydroxylamine of cystathionine-β-synthase (CBS), aggravated and remitted the hypoxic damage in the cortex cells, respectively. In addition, treatment with EA significantly downregulated the expression of nNOS and NF-κB in the rat cortex cells (*P<0.05, **P<0.01, compared with the control groups). The results also indicated that treatment with EA downregulated the NO content of cortical cells against HIBD via the NF-κB/nNOS pathway and further implied that the hydrogen sulfide/CBS system may be involved in the process. The present study provided a significant reference for the prevention and treatment of HIBD using the EA technique and also described a novel protective mechanism.