Background and Purpose: Mitochondrial fission-fusion dynamics and bioenergy dysfunctions are participated in cerebral ischemia/reperfusion (I/R) injury. Our study aims to investigate the role of Mul1-dependent Mfn2 ubiquitination and its mediated mitochondrial dysfunctions and explain the molecular mechanism of ginsenoside compound K (CK) targeting Mul1 against cerebral I/R injury. Experimental Approach: We used a combination of in vitro and in vivo models, including oxygen and glucose deprivation/reperfusion-induced PC12 cell model and middle cerebral artery occlusion/reperfusion-induced rat model, to mimic I/R injury. The potential mechanisms and pharmacological effects of ginsenoside CK on mitochondrial dynamics and bioenergy were evaluated by Mul1 knockdown and pharmacological antagonism study using a series of experiments. Key Results: I/R injury stimuli upregulated the binding of Mul1 with Mfn2 to regulate Mfn2 ubiquitination and degradation, which resulted in increased mitochondrial fission, bioenergy dysfunction, neuronal apoptosis, and neurological impairment. Knockdown of Mul1 exerted beneficial effect on cerebral I/R-induced neuronal death by abolishing mitochondrial fission, mitophagy, and bioenergy dysfunction. More importantly, ginsenoside CK mainly inhibited Mul1 expression to reduce Mfn2 ubiquitination and mitochondrial translocation of DRP1, thereby inhibiting mitochondrial fission, mitophagy and mitochondrial apoptosis against cerebral I/R injury in both in vitro and in vivo models. Conclusions and Implications: These data for the first time explain molecular basis of the Mul-dependent mitochondrial dysfunctions during I/R damages and provide the evidence that ginsenoside CK may be a promising therapeutic agent against cerebral I/R injury by targeting Mul1/Mfn2-mediated mitochondrial dynamics and bioenergy.