Mitochondrial defects are early pathological changes in neurodegenerative disease (ND). Homocysteine (Hcy) is an independent risk factor for ND. However, whether and how Hcy induces mitochondrial defects during the process of neurodegeneration is unclear. Here, we revealed that Hcy interfered with mitochondrial oxidative phosphorylation (OXPHOS) by inhibiting the mitochondrial electron transport chain (ETC) complex I, resulting in increased levels of reactive oxygen species (ROS) in the hippocampus of rats. Specifically, Hcy suppressed Ndufa1 expression, which is essential for complex I assembly and activation, by interfering with its transcription factor Creb1. Moreover, we found that Hcy induced neurodegeneration-like pathological changes in mitochondria in the brain via the inhibition of the NAD/Sirt1 pathway, including defects in mitochondrial morphology, mitochondrial biogenesis, and mitophagy, ultimately leading to impairments in synapses and cognition, all of which were reversed by Ndufa1 upregulation. Thus, Ndufa1 is a key molecular switch of Hcy-induced mitochondrial damage, and appropriately targeting Ndufa1 or NAD replenishment may serve as a novel therapeutic strategy for Hcy-induced neurodegeneration and cognitive impairment.