To employ bioinformatics to screen for neuroprotective drugs for cerebral ischemia and evaluate them in an animal transient Middle Cerebral Artery Occlusion (tMCAO) model. Methods: Two datasets from the Gene Expression Omnibus (GEO) database, representing the 6 h and 12 h time points after MCAO, were obtained and subjected to differential gene analysis. The biological processes and signaling pathways of the differentially expressed genes were enriched using DAVID and Metascape. Key genes were identified using Cytoscape and validated in a mouse tMCAO model. The Connectivity Map (Cmap) online tool was used to predict potential therapeutic drugs for cerebral ischemia, and the autophagy inhibitor KU-0063794 was selected to investigate its neuroprotective effect on tMCAO mice. Results: A total of 289 common differentially expressed genes were identified between the two datasets. Enrichment analysis revealed that the differentially expressed genes were primarily enriched in biological processes such as regulation of synaptic transmission, lipid biosynthesis, and apoptosis during the acute phase of ischemic stroke. Real-time PCR results showed that the mRNA levels of Hmox1, Serpine1, Ptgs2, and Timp1 in the peri-infarct cortex increased 12 h after tMCAO. Western blotting results indicated that the expression level of Hmox1 increased from 6 to 24 h after ischemic injury. KU-0063794 significantly reduced cerebral infarct volume in tMCAO mice, mitigated oxidative stress and neuronal apoptosis by activating the Nrf2/Hmox1 pathway, and behavioral tests demonstrated that KU-0063794 significantly improved motor dysfunction in tMCAO mice. Conclusion: KU-0063794 exerts neuroprotective effects by reducing oxidative stress and neuronal apoptosis.