Aim To observe whether the Wnt/β-catenin signaling pathway mediates the protective effect of Shenmai injection (SM) on cerebral ischemia-reperfusion injury (CIRI) in rats. Methods According to the randomized block principle, 84 SD rats were divided into 7 groups:sham operation group (sham), model group (MCAO), SM＋model group (SM＋MCAO), normal saline (NS)＋model group (NS＋MCAO), phosphate buffered solution (PBS)＋SM＋model group (PBS＋SM＋MCAO), Wnt/β-catenin antagonist dickkopf-1 (Dkk-1)＋SM＋model group (Dkk-1＋SM＋MCAO), Dkk-1＋model group (Dkk-1＋MCAO), 12 rats in each group. Rat CIRI model was established by middle cerebral artery occlusion (MCAO) with line embolus. Immediately after cerebral ischemia, SM＋MCAO group, PBS＋SM＋MCAO group and Dkk-1＋SM＋MCAO group were intraperitoneally injected with SM (15 mL/kg), whereas NS＋MCAO group was injected with equal amount of NS. At 30 min before the model was set up, Dkk-1＋SM＋MCAO group and Dkk-1＋MCAO group were injected with Dkk-1 (1 g/L, 1 μL) to the ventricle, whereas PBS＋SM＋MCAO group was injected with equal amount of PBS. After reperfusion for 24 h, neurobehavioral score was assessed in each group, cerebral infarction volume was measured, expressions of Bcl-2, Bax and β-catenin protein were detected in the semi-dark zone of cerebral infarction, and Bcl-2/Bax ratio was calculated. Results Compared with the sham group, neurobehavioral score, cerebral infarction volume, and the expressions of Bcl-2, Bax, and β-catenin proteins were increased in the MCAO group (P＜0.05), and Bcl-2/Bax ratio was reduced (P＜0.05). Compared with the MCAO group, neurobehavioral score, cerebral infarction volume and Bax protein expression were significantly reduced in the SM＋MCAO group (P＜0.05), the expressions of Bcl-2 and βcatenin proteins and Bcl-2/Bax ratio were significantly increased (P＜0.05). Compared with the SM＋MCAO group, neurobehavioral score, cerebral infarction volume and Bax protein expression were significantly increased in the Dkk-1＋SM＋MCAO group (P＜0.05), while Bcl-2 protein expression and Bcl-2/Bax ratio were significantly decreased (P＜0.05). Conclusion Shenmai injection improves brain damage of CIRI rat by up-regulation of Wnt/β-catenin signaling pathway.