: To investigate whether the neuroprotective effect of tissue kallikrein (TK) on ischemic stroke is related to the induction of protective autophagy by in vivo experiments. Methods: Adult male SD rats were randomly divided into sham surgery group, normal saline (NS) group, TK group, 3-MA group and 3-MA+ TK group, with 11 rats in each group. Rats in the sham group and the NS group were injected NS in the lateral ventricle, rats in the TK group and in the 3-MA group were injected TK and autophagy inhibitor 3-MA in the lateral ventricle respectively, and rats in the 3-MA+TK group were injected with 3-MA and then injected with TK 30 min later. Permanent middle cerebral artery occlusion (pMCAO) model was established by thread embolism after the lateral ventricle administration was completed in each group. At 12 h post-ischemia, the neurological function of rats in each group was assessed by Longa score. After the completion of nerve injury assessment, the cerebral infarction volume of rats in each group was detected by TTC staining, and the expression levels of autophagy related proteins LC3 and p62 were detected by western blotting. Results: The expression of LC3-Ⅱ in ischemic cortex of NS group was higher than that in sham group, and the expression of p62 was lower (both P< 0.01). Compared with the NS group, the expression of LC3-II in the ischemic side cerebral cortex of the TK group was increased, the expression of p62 decreased, the neurological damage of rats was milder, and the volume of cerebral infarction was significantly reduced (all P<0.01). Compared with the TK group, the expression of ischemic cerebral cortex LC3-II and the expression level of p62 were higher in the 3-MA group, and the rats had more severe neurological damage and larger cerebral infarction volume (all P<0.01). Compared with the TK group, the score of nerve damage and the volume of cerebral infarction increased significantly in the 3-MA+TK group (all P<0.01). Conclusion: TK has a neuroprotective effect on ischemic stroke rats, and its mechanism is related to the induction of autophagy genesis.