AIM: To determine whether Jiunaoning injection has protective effects on oxygen/glucose-deprived and reperfusion injury-induced neurons. METHODS: Various concentrations of Jiunaoning injection (0.5-5 mL/L) were used to observe their effects on cultured rat cortical neurons induced by oxygen/glucose-deprived and reperfusion injury in various time points. The neuronal metabolic rate and viability were assessed by using 3-(4,5-dimethylthiazol)-2, 5-diphenyl-tetra zoliumbromide (MTT) and lactate dehydrogenase (LDH) assay. RESULTS: Jiunaoning injection enhanced the neuronal metabolic rate in a dose-dependent manner in the range from 0.5 to 5 mL/L, and Jiunaoning injection (1.5-2.5 mL/L) enhanced the neuronal metabolic rate, decreased the cell death rate and depressed LDH leak rate significantly. CONCLUSION: Jiunaoning injection has an affirmative protective effect on oxygen/glucose-deprived and reperfusion-induced neuronal injury.