BACKGROUND AND PURPOSE: The role of immune checkpoint B7-H3 in acute ischemic stroke prognosis and post-stroke immunosuppression remains uninvestigated, despite the clinical significance of immune checkpoints with inflammaging and post-stroke infections. In this study, we investigated the effect of regulating the cerebral induction of B7-H3 after acute ischemic stroke, evaluated its effect on brain damage, neuroinflammation, vascular integrity, host defense gene regulation, and functional outcomes. METHODS: C57BL/6 mice were subjected to transient middle cerebral artery occlusion and injected (i.v.) with either B7-H3 siRNA or a negative (non-targeting) siRNA at 5 min after reperfusion. At 24 hours of reperfusion, magnetic resonance imaging (MRI) of the mouse brain was performed using a 9.4 T scanner to assess brain damage (T2, ADC, and Kurtosis). Real-time qPCR and NanoString nCounter Neuroinflammation Panels were used to determine the acute changes in overall neuroinflammatory functions that are mediated by B7-H3. Motor function (beam walk, rotarod tests, and grip strength) was assessed between days 1 and 7 of reperfusion. RESULTS: Early inhibition of B7-H3 after stroke significantly reduced the brain damage and promoted the functional outcomes. Post-stroke neuroinflammation was reprogrammed with B7-H3 inhibition towards balancing of neuroprotective anti-inflammatory mechanisms without compromising the immune response that is crucial for preventing post-stroke infections. CONCLUSIONS: Our results demonstrate that the induction of B7-H3 during the acute period after stroke is a mediator of post-stroke neuroinflammation and secondary brain damage.