This study aimed to investigate the role of nitric oxide (NO) in the neuroprotective effects of helium preconditioning (He-PC) in a neonatal hypoxia/ischemia (HI) rat model. Seven-day old rat pups were divided into normal control group, He-PC group, HI group, He-PC + HI group, L-NAME + HI group and L-NAME + He-PC + HI group. HI was induced by exposure to 80% oxygen for 90 min. He-PC was conducted with 70% helium—30% oxygen for three 5-min periods. Three hours after He-PC, animals in control group and He-PC group were sacrificed, and the brain was collected for the detection of NO content. At 24 h after HI, animals in control group, HI group, He-PC + HI group, and L-NAME + He-PC + HI group were sacrificed, and the brain was collected for detection of infarct ratio, antioxidases (SOD, HO-1 and Nrf2), DNA binding activity of Nrf2 and TUNEL staining. Three weeks later, the neurological function and brain atrophy were determined. Results showed pretreatment with L-NAME alone failed to exert protective effect on HI. He-PC significantly increased NO content, reduced the brain infarct area, increased anti-oxidases expression and DNA binding activity of Nrf2, decreased the apoptotic cells, and improved the neurological function and brain atrophy. In addition, this protection was markedly inhibited by L-NAME (a non-selective NOS inhibitor). These findings suggest that the He-PC may induce NO production to activate Nrf2, exerting neuroprotective effect on neonatal HI.