Glucose fluctuations have an adverse effect on several diabetes-related complications, especially for the nervous system, but the underlying mechanisms are not clear. MicroRNAs are critical regulators of posttranscription in many physiological processes, such as apoptosis. Our study clarified the neuroprotective effects of miR-129-3p targeting mitochondrial calcium uniporter (MCU) in glucose fluctuation-mediated neuronal damage and the specific mechanisms involved. The expression of MCU and miR-129-3p was examined by real-time PCR and Western blot in the glucose fluctuation cell model. Dual-luciferase reporter assay was performed to confirm the transcriptional regulation of miR-129-3p by MCU. Fluorescent probe and assay kit assay was used to determine oxidative stress condition. Mitochondrial-dependent intrinsic apoptotic factors were examined by flow cytometry assay, enzyme-linked immunosorbent assay (ELISA), and gene and protein expression assays. We found an upregulation of MCU and downregulation of miR-129-3p in glucose fluctuation-treated primary hippocampal neuronal cells, and miR-129-3p directly targeted MCU. miR-129-3p overexpression produced a dramatic reduction in calcium overload, reactive oxygen species (ROS) generation, GSH-to-GSSG ratio, MMP-2 expression in the mitochondrial-dependent intrinsic apoptosis pathway and an increase in MnSOD activity. Increasing MCU expression rescued the effects of miR-129-3p overexpression. miR-129-3p downregulation produced a significant increase in calcium overload, reactive oxygen species (ROS) generation, MMP-2 expression, cytochrome c release and cell apoptosis, and antioxidant N-acetyl cysteine (NAC) rescued the effects of miR-129-3p downregulation. Therefore, miR-129-3p suppressed glucose fluctuation-mediated neuronal damage by targeting MCU via a mitochondrial-dependent intrinsic apoptotic pathway. The miR-129-3p/MCU axis may be a promising therapeutic target for glucose fluctuation-mediated neuronal damage. © 2021 Wang et al.