In order to evaluate the thrombolytic effect of Recombinant staphylokinase for injection (r-SaK) in acute ischemic cerebral infarction, an intracranial large vessel occlusion animal model was generated by pushing an autologous thrombus to the internal carotid artery under X-ray angiography. Autologous thrombi/saline were injected into the internal carotid artery, and thrombolytic agents were then administrated. Thrombus formation and dissolution were monitored by real-time digital subtraction angiography (DSA), blood coagulation and histopathologic examinations were used as subsidiary methods. The results in the present study showed that the left cerebral vascular thrombotic occlusion model was established stably after the autologous thrombus pushed through the internal carotid artery in dogs. Administration of r-SaK (0.25, 0.5, 1.0 mg/kg) produced effective thrombus dissolution with a recovery of over 80% blood flow, as effective as alteplase (1.68 mg/kg). Correspondingly, blood coagulation was changed by r-SaK, with a dramatic elongation of prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time (TT) and reduction of fibrinogen (FIB). In contrast to the model group, pathological improvement in the two thrombolytic groups were mainly manifested in the improvement of the structural integrity of the gray matter, and the reduction of the infiltration of inflammatory cells and neuronal damage in the intracranial blood vessels. Besides, no adverse reactions related to bleeding in this model were found. The results indicate that intravenous infusion of r-SaK has a significant thrombolytic effect on intracranial large vessel occlusion model, and can prevents brain tissue and neuron damage induced by thromboembolism.