The aim is to investigate the effects of neuregulin-1β (NRG-1β) on expression of matrix metalloproteinase-9 (MMP-9) and neuron-specific enolase (NSE) in brain tissue in rats following cerebral ischemia/reperfusion. One hundred and fifty adult healthy male Wistar rats were used in the present study. Ten of them were randomized into a sham-operation group (n′=′10) and the rest suffered surgery operation of middle cerebral artery occlusion/reperfusion with intraluminal monofilament suture from the left external–internal carotid artery. As a result, 100 rats of successful models were randomly divided into a control group (n′=′50) and a treatment group (n′=′50). Rats in the treatment group were injected 1.5% NRG-1β at a dosage of 0.3 μg/kg from the stump of the left external carotid artery into the internal carotid artery. The expressions of MMP-9 and NSE proteins were determined by immunohistochemical, immunofluorescent double labeling, and Western blot assay. Ischemia/reperfusion induced morphological changes of brain tissue, including neurocyte shrinkage, chromatin condensation, nuclei fragment, and gliacyte and endothelial cell swelling. NRG-1β obviously reduced and delayed the cerebral damage. With the duration of ischemia, the expression of MMP-9 gradually increased in the control group. NRG-1β decreased the level of MMP-9 compared with that in the control group (P′<′0.01). NSE immunoreaction transiently elevated at the early stage of cerebral ischemia insult, and then gradually decreased in the control group. The administration of NRG-1β significantly increased the level of NSE, and thus delayed the time and the degree of neuron damage. There were statistical differences in contrast to the control group (P′<′0.01). There was no relationship between the expressions of the two proteins. MMP-9 might aim at various target cells at different stages and contribute to the inflammatory reaction after cerebral ischemia–reperfusion insult. NRG-1β inhibits the activation of MMP-9 and development of inflammation, enhances the activity of NSE, improves the microenvironment of neuron survivals, and delays the phase of irreversible neuron necrosis. Therefore, NRG-1β may play a neuroprotective role in cerebral ischemia/reperfusion.