<p>Mitochondrial calcium homeostasis may offer therapeutic benefits for ischemic stroke. Harpagide has been shown to inhibit mitochondrial calcium uptake, but the mechanism remains unclear. In this study, a male ICR mouse model of permanent middle cerebral artery occlusion (pMCAO)-induced focal cerebral ischemia and PC12 and SH-SY5Y cells exposed to oxygen–glucose deprivation (OGD) were used. Apoptosis, lactate dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA), and the ATP levels were measured to assess the neuroprotective effects of harpagide. DCFH-DA, JC-1, and Rhod-2A probes were used to analyze the mitochondrial function. RT-qPCR and western blot were used to determine the mitochondrial calcium uniporter (MCU), the apoptosis-inducing factor (AIF), Endo G, Cyt C, Caspase-3, and AMPK–MCU phosphorylation expression levels. Additionally, Longa scoring and triphenyl tetrazolium chloride (TTC) staining were used. Transmission electron microscopy (TEM) was used to examine the mitochondrial ultrastructural changes. An immunohistochemical analysis was conducted to detect the MCU expression in the temporal cortex. Harpagide attenuated OGD-induced cytotoxicity, as evidenced by reduced lactate LDH leakage, restored ATP generation, and ameliorated mitochondrial ultrastructural damage. Mechanistically, harpagide suppressed oxidative stress and alleviated mitochondrial calcium ([Ca²⁺]_mito) overload, concomitant with modulated expression of MCU complex components and apoptosis-related proteins. These neuroprotective effects were linked to harpagide-mediated AMPK expression and subsequent preservation of mitochondrial bioenergetics and calcium homeostasis. Collectively, these findings position harpagide as a promising phytochemical candidate for ameliorating mitochondrial dysfunction in ischemic stroke, warranting further investigation into its precise molecular interactions with the AMPK–MCU axis.</p>