Cerebral ischemia causes blood-brain barrier (BBB) injury and thus increases the risk of complications secondary to thrombolysis, which limited its clinical application. This study aims to clarify the role and mechanism of salidroside (SALD) in alleviating brain ischemic injury and whether pretreatment of it could improve prognosis of delayed treatment of tissue plasminogen activator (t-PA). Rats were subjected to 3 h of middle cerebral artery occlusion (MCAO) and were intraperitoneally administered with 10, 20 or 40 mg/kg SALD before ischemia. 1.5% 5-triphenyl-2H-tetrazolium chloride (TTC) staining and neurological studies were performed to observe the effectiveness of SALD. The expressions and the distribution of phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) signaling were analyzed. Experiments were further conducted in isolated microvessels and human brain microvascular endothelial cells (HBMECs) to explore the protective mechanism of SALD. Finally, rats were subjected to 6 h of MCAO and 24 h of reperfusion. tPA was given with or without the pretreatment of SALD. Various approaches including gelatin zymography, western blot and immunofluorescence were used to evaluate the effect of this combination therapy. SALD could reduce cerebral ischemic injury and enhance HBMECs viability subjected to OGD. In vivo and in vitro studies showed the mechanism might be related to the activation of PI3K/Akt signaling by phosphorylating Akt on Ser473. Pretreatment of SALD could alleviate BBB injury and improve the outcome of delayed treatment of tPA. These results provide evidence that SALD might be an effective adjuvant to reduce the complications induced by delayed tPA treatment for brain ischemia.