To investigate the relationship between thymosin β4 (Tβ4) expressed in microglia and the phosphatidylinositol-3-kinase (PI3K)/kinase B (AKT) signaling pathway after focal brain ischemia/reperfusion injury (I/R). Methods Focal cerebral I/R injury models were established in rats, and oxygen-glucose deprivation/reperfusion (OGD/R) was established in the immortalized mouse microglial cell line BV2. Immunohistochemistry was used to detect the level of Tβ4 in the cerebral cortex and BV2 cells. Furthermore, by interference Tβ4 gene expression using shRNA, changes in the levels of key PI3K/AKT pathway proteins were detected by Western blotting. Results The results showed that after focal I/R injury in rats, especially after I 2 h/R 48 h, Tβ4 levels in the cerebral cortex were significantly increased (P＜0.0001 ). Further study confirmed that the Tβ4 expression significantly increased in BV2 cells after OGD/R compared with the control group, especially the OGD 6 h/R 48 h group (P＜0.0001), and the p-PI3K/PI3K and p-AKT/AKT ratio also increased (P＜0.001). However, when Tβ4 gene was interfered, the p-PI3K/PI3K (P＜0.05 ) and p-AKT/AKT (P＜0.01 ) ratios were significantly inhibited. Conclusion: Tβ4 is highly expressed in cortical microglia after focal brain I/R injury. The high Tβ4 expression in microglia may influence the repair of cerebral ischemia/reperfusion injury by the PI3K/AKT pathway.