Cerebral ischemia-reperfusion injury (CIRI) has emerged as a hindrance for rehabilitation of ischemic stroke patients. Naotaifang (NTF) exhibits beneficial efficacy in alleviating inflammation and ferroptosis in vitro during CIRI. While the potential role of NTF in regulating mitochondrial dynamics in CIRI are not elucidated. This study aimed to explore the mechanism of NTF against CIRI by regulating the dynamin-related protein 1 (Drp1)-dependent mitochondrial fission/fusion. Modeling middle cerebral artery occlusion/reperfusion (MCAO/R) in vivo to evaluate the effects of NTF on the MCAO/R-damaged neurons and the structure, dynamics and function of mitochondria. An oxygen-glucose deprivation/reperfusion (OGD/R) cell model was established to evaluate the role of NTF in OGD/R-damaged cells. Function of Drp1 in CIRI and the neuroprotection of NTF through the mitochondrial fission/fusion pathway were investigated in vivo and in vitro. The results revealed that in vivo, NTF alleviated neuron injury in a dose-dependent manner, down-regulated Drp1 and fission protein 1 (Fis1) levels, upregulated optic atrophy 1 (Opa1), mitofusin 1/2 (Mfn1 and Mfn2), facilitated mitochondrial fusion and inhibited mitochondrial fission to rescue cells from CIRI. In vitro, Drp1 overexpression inhibited mitochondrial fusion and activated mitochondrial fission, while silencing of Drp1 exhibited the opposite result. NTF rebalanced mitochondrial dynamic in the OGD/R cell model. NTF could alleviate neuron injury following CIRI by regulating the balance of mitochondrial fission and fusion. Targeting Drp1-dependent mitochondrial dynamics may represent a viable treatment strategy for addressing the issues of CIRI post ischemic stroke.