Oxysophoridine, a new alkaloid extracted from Sophora alopecuroides L., has been shown to have a protective effect against ischemic brain damage. In this study, a focal cerebral ischemia/reperfusion injury model was established using middle cerebral artery occlusion in mice. Both 62.5, 125, and 250 mg/kg oxysophoridine, via intraperitoneal injection, and 6 mg/kg nimodipine, via intragastric administration, were administered daily for 7 days before modeling. After 24 hours of reperfusion, mice were tested for neurological deficit, cerebral infarct size was assessed and brain tissue was collected. Results showed that oxysophoridine at 125, 250 mg/kg and 6 mg/kg nimodipine could reduce neurological deficit scores, cerebral infarct size and brain water content in mice. These results provided evidence that oxysophoridine plays a protective role in cerebral ischemia/reperfusion injury. In addition, oxysophoridine at 62.5, 125, and 250 mg/kg and 6 mg/kg nimodipine increased adenosine-triphosphate content, and decreased malondialdehyde and nitric oxide content. These compounds enhanced the activities of glutathione-peroxidase, superoxide dismutase, catalase, and lactate dehydrogenase, and decreased the activity of nitric oxide synthase. Protein and mRNA expression levels of N-methyl-D-aspartate receptor subunit NR1 were markedly inhibited in the presence of 250 mg/kg oxysophoridine and 6 mg/kg nimodipine. Our experimental findings indicated that oxysophoridine has a neuroprotective effect against cerebral ischemia/reperfusion injury in mice, and that the effect may be due to its ability to inhibit oxidative stress and expression of the N-methyl-D-aspartate receptor subunit NR1.