Background <p>Chronic stress contributes to hippocampal neuronal apoptosis and neural circuit dysfunction in major depressive disorder (MDD), but the epitranscriptomic mechanisms regulating stress-responsive microRNAs remain unclear. This study investigated whether methyltransferase-like 3 (METTL3)-mediated N6-methyladenosine (m6A) modification promotes miR-140-3p maturation and neuronal apoptosis through the OTX2/Wnt/β-catenin axis under chronic stress.</p> Methods and results <p>A chronic unpredictable mild stress rat model and a corticosterone-induced primary hippocampal neuron injury model were established. Behavioral assays, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, flow cytometry, western blotting, qRT-PCR, proteomic and transcriptomic analyses, methylated RNA immunoprecipitation (MeRIP), DiGeorge syndrome critical region gene 8 (DGCR8) RNA immunoprecipitation (RIP), and dual-luciferase reporter assays were performed. Serum miR-140-3p expression was also assessed in patients with MDD and healthy controls. miR-140-3p was upregulated in the hippocampus of stressed rats and in the serum of patients with MDD. Hippocampal miR-140-3p knockdown alleviated depressive-like behaviors and reduced neuronal apoptosis, whereas miR-140-3p inhibition protected primary hippocampal neurons from corticosterone-induced apoptosis. Mechanistically, chronic stress increased METTL3 expression and global m6A levels. METTL3 enhanced m6A modification of pri-miR-140-3p, promoted DGCR8-dependent processing, and increased mature miR-140-3p expression. miR-140-3p directly targeted OTX2 and modulated Wnt/β-catenin signaling, while OTX2 silencing attenuated the antiapoptotic effect of miR-140-3p inhibition.</p> Conclusions <p>METTL3-dependent m6A maturation of miR-140-3p may contribute to stress-induced hippocampal neuronal apoptosis through the OTX2/Wnt/β-catenin axis, providing potential molecular targets for further investigation in MDD.</p>