Natural bioactive compounds have increasingly proved to be promising in evidence- or target-directed treatment or modification of a spectrum of diseases including cerebral ischemic stroke. Hydroxysafflor yellow A (HSYA), a major active component of the safflower plant, has drawn more interests in recent year for its multiple pharmacological actions in the treatment of cerebrovascular and cardiovascular diseases. Although the Janus kinase signaling, such as JAK2/STAT3 pathway, has been implicated in the modulation of the disease, the inhibition or activation of the pathway that contributed to the neuronal prevention from ischemic damages remains controversial. In this study, a series of experiments were performed to examine the dose- and therapeutic time window-related pharmacological efficacies of HSYA with emphasis on the HSYA-modulated interaction of JAK2/STAT3 and SOCS3 signaling in the MCAO rats. We found that HSYA treatment significantly rescued the neurological and functional deficits in a dose-dependent manner in the MCAO rats within 3 h after ischemia. HSYA treatment with a dosage of 8 mg/kg or higher markedly downregulated the expression of the JAK2-mediated signaling that was activated in response to ischemic insult, while it also promoted the expression of SOCS3 coordinately. In the subsequent experiments with the use of the JAK2 inhibitor WP1066, we found that the treatment of WP1066 alone or combination of WP1066/HSYA all exhibited inhibitory effects on JAK2-mediated signaling, while there was no influence on the SOCS3 activity of corresponding efficacious data in the MCAO rats, suggesting that excessive activation of JAK2/STAT3 might be necessary for HSYA to provoke SOCS3-negative feedback signaling. Taking together, our study demonstrates that HSYA might modulate the crosstalk between JAK2/STAT3 and SOCS3 signaling pathways that eventually contributed to its therapeutic roles against cerebral ischemic stroke.