To explore the protective mechanism of calycosin on cerebral ischemia reperfusion injury in rats. Methods: Eighty SD rats were randomly divided into the groups sham operation, ischemia/ reperfusion (I/R), low-dose, medium-dose and high-dose. Fourteen days before model establishment, the rats of low-dose, middle-dose, and high-dose groups were intraperitoneally injected with 5 mg/(kg·d), 10 mg/(kg·d), and 20 mg/(kg·d) calycosin, respectively; the sham and I/R group rats were injected with an equal amount of dimethyl sulfoxide. Next the cerebral ischemia and reperfusion models were constructed. Twenty-four hours later, the neurological function score of rats was evaluated, the cerebral infarction volume and brain water content were calculated, and the superoxide dismutase (SOD) and malondialdehyde (MDA) content and NF-κB expression in the brain tissue were determined. Results: The neurological function score, cerebral infarction volume, and brain water content of the low-dose, medium-dose, and high-dose groups decreased with the increase of calycosin concentration, with the high-dose group showing the most significant decrease (P<0.05 or P<0.01), and all showed lower values than the I/R group (P<0.05 or P<0.01). The SOD activity in the brain tissue of the low-dose, medium-dose, and high-dose groups was significantly higher than that of the I/R group (P< 0.01), and the MDA level was significantly lower than that of the I/R group (P<0.01); the SOD activity of the low-dose, medium-dose, and high-dose groups increased with the increase of calycosin concentration (P<0.05 or P<0.01); the MDA level of the low-dose, medium-dose, and high-dose groups decreased with the increase of calycosin concentration (P<0.05 or P<0.01). Compared with the I/R group, the expression of NF-κ B in the low-dose, medium-dose, and high-dose groups was significantly down-regulated (P<0.01); the expression of NF-κB in the low-dose, medium-dose, and high-dose groups was down-regulated with the increase of calycosin concentration, with the high-dose group showing the most significant down-regulation (P<0.05 or P<0.01). Conclusion: Calycosin has protective effects in cerebral ischemia reperfusion rats.