It was previously shown that SHPL-49, a glycoside derivative of salidroside formed through structural modification, exhibited neuroprotective effects in a rat cerebral ischemia model of permanent middle cerebral artery occlusion (pMCAO). Additionally, SHPL-49 enhanced the mRNA expression of vascular endothelial growth factor-a ( Vegf-a ) in macrophages. Microglia, functioning as resident macrophages within the brain, promptly respond to cerebral ischemia and engage in interactions with the cells of the Glial-Vascular Unit to orchestrate nerve injury responses. We postulated that the neuroprotective effects of SHPL-49 were mediated through microglia-dependent amelioration of endothelial dysfunction following cerebral ischemia. The present study demonstrates that SHPL-49 effectively mitigated microglia-dependent endothelial dysfunction in the pMCAO model by upregulating the expression of VEGF and suppressing the release of MMP-9 from microglia. Further MRI analyses confirmed that SHPL-49 significantly reduced nerve and endothelial function when microglia were depleted in the brains of pMCAO rats. The above phenomenon was also confirmed in the in vitro experiment investigating microglia-mediated brain endothelial cell function. Furthermore, we discovered that SHPL-49 activates the VEGFR2/Akt/eNOS pathways in endothelial cells and suppresses the p38 MAPK/MMP-9 pathways in microglia cells, thereby facilitating brain endothelial cell protection. Altogether, we have demonstrated that SHPL-49 effectively ameliorates endothelial dysfunction induced by cerebral ischemia through a microglia-dependent mechanism, thereby providing more valuable insights and references for the clinical evaluation of SHPL-49 injection for ischemic stroke.