This study was undertaken to find out the effects of acetylpuerarin on hippocampal neurons and intracellular free calcium in primary culture subjected to oxygen-glucose deprivation/reperfusion. Methods: According to different reperfusion time (1h, 6h, 12h, 24h), three concentrations (1.6 μmol l−1, 0.4 μmol l−1, 0.1 μmol l−1) of acetylpuerarin, and MK-801 (10 μmol l−1), a positive control drug, neurons were randomly divided into 21 groups. Each group was observed by inverted phase contrast microscope; neuron viability was measured by the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT); intracellular Ca2+ was observed by Fura-2/AM ester through fluorospectrophotometer. Results: The injured neurons were protected and degeneration and necrosis were alleviated in treatment groups of acetylpuerarin and MK-801. Acetylpuerarin increased the neuron viability at high, middle and low concentrations. Fluorescence detection results showed that the calcium concentration in the group treated with acetylpuerarin and MK-801 was lowered in each reperfusion time. Conclusion: Our results demonstrated that acetylpuerarin could protect the hippocampal neurons from ischemia-reperfusion injury in rats by alleviating the morphological damage, increasing neuron viability and decreasing calcium concentration in neuron.