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BACKGROUND: Acupuncture improves the prognosis of neonatal hypoxic-ischemic encephalopathy (HIE). However, the cytological mechanism of acupuncture therapy remains poorly understood. In situ neural stem cell (NSC) proliferation theory proposes that the proliferation and differentiation of NSCs plays an important role in HIE treatment with acupuncture. OBJECTIVE: To investigate NSC proliferation and differentiation in the brain of a rat model of HIE during acupuncture at Ren, Du, and urinary bladder meridians. DESIGN, TIME AND SETTING: A randomized, controlled animal experiment was performed at the Central Laboratory of Shantou University Medical College from July 2005 to June 2009. MATERIALS: A 32~# 1-cun stainless steel acupuncture needle was purchased from Suzhou Acupuncture Supplies Co. Ltd., China. METHODS: A total of 90 Sprague-Dawley rats, aged 7 days, were randomly assigned to acupuncture, model and normal groups, with 30 animals in each group. Animals in acupuncture and model groups were subjected to left common carotid artery ligation followed by hypoxia for 2 hours to establish neonatal HIE models. Acupuncture group rats underwent acupuncture at Ren, Du, and urinary bladder meridians, once a day. MAIN OUTCOME MEASURES: The number, appearance, and distribution of bromodeoxyuridine (BrdU)-positive cells in the cerebral cortex and hippocampus of each group were compared. In addition, NSC differentiation in the occipital cortex and hippocampal dentate gyrus 40 days following model establishment was detected. RESULTS: BrdU-positive cells were dispersed in the cerebral cortex and hippocampus. The number of BrdU-positive cells in occipital cortex and hippocampal dentate gyrus of HIE rats remained unchanged following 3 and 7 days of acupuncture, but a significant increase was detected on days 14 and 28 (P < 0.01 or P < 0.05). At 40 days, immunofluorescence showed that a majority of BrdU-positive cells were co-lableled with the neuron marker, and neuron specific enolase, and a few were co-labeled with the astrocyte marker, and glial fibrillary acidic protein. CONCLUSION: Acupuncture at Ren, Du, and urinary bladder meridians promoted NSC proliferation in the occipital cortex and hippocampal dentate gyrus of HIE rats. Moreover, acupuncture-induced neoformative NSCs mostly differentiated into neurons.