To study the effect and protective mechanism of Naotaitong Formula in the rat model of focal cerebral ischemia-reperfusion injury.Methods:Rats were modeled for focal cerebral ischemia-reperfusion injury and then randomized into model control，positive control，Naotaitong，and drug combination groups.Healthy SD rats were selected as the sham operation group.The National Institute of Health stroke scale(NIHSS) score and the water content and infarct volume percentage in the brain tissue were evaluated and determined.The immunohistochemical method was employed to measure the average optical density values of B-cell lymphoma-2(Bcl-2) and Bcl-2-associated X protein(Bax).Western blotting and RT-PCR were employed to determine the protein and mRNA levels of forkhead box O3a(FoxO3a)，hypoxia-inducible factor 1 alpha(HIF-1α)，nuclear factor-kappa B(NF-κB)，brain-derived neurotrophic factor(BDNF) in the brain tissue.Results:Compared with the sham operation group，the modeling increased the NIHSS score，water content and infarct volume percentage in the brain tissue，the protein and mRNA levels of FoxO3a，HIF-1α，NF-κB，and BDNF in the brain tissue，the average optical density values of Bax and Bcl-2，and the Bax/Bcl-2 ratio(P<0.05).Compared with the model control group，drug interventions reduced the NIHSS score，water content and infarct volume percentage in the brain tissue，the average optical density of Bax，the Bax/Bcl-2 ratio，and the protein and mRNA levels of NF-κB in the brain tissue(P<0.05)，and the drug combination group showed the greatest reduction(P<0.05).In addition，the average optical density of Bcl-2，the protein and mRNA levels of FoxO3a，HIF-1α，and BDNF were increased(P<0.05)，and the drug combination group had the greatest increase(P<0.05).Conclusion:Naotaitong Formula can protect the brain tissue from focal cerebral ischemia injury in the rat model by regulating the FoxO3a/HIF-1α/NF-κB signaling pathway.