<p>Slow transit constipation (STC) is a prevalent functional gastrointestinal disorder characterized by a reduced frequency of bowel movements, the presence of dry and hard stools, and abdominal pain. However, the underlying mechanisms contributing to its pathogenesis have not yet been fully clarified. This study aims to investigate the effects of METTL3 on loperamide (LOP)-induced STC mice and glutamic acid-induced interstitial cells of Cajal (ICCs). METTL3-knock down adeno-associated virus (AAV) was used to treat LOP-induced mice, and the effect of METTL3 down-regulation was assessed by the stool parameters, histological analysis, transmission electron microscopy (TEM), TdT-mediated dUTP nick end labeling (TUNEL) staining, immunohistochemistry, Immunofluorescence staining, and Western blotting. METTL3 small interfering RNA (siRNA) was transfected into ICCs before glutamic acid, PI3K inhibitor (LY294002), and AKT inhibitor (GSK690693) treatment alone or in combination. EdU assays, flow cytometry, TEM, and Western blot were used to investigate the relationship between METTL3 and PI3K/AKT pathway. METTL3 deletion alleviated constipation symptoms and promoted intestinal motility in STC mice. METTL3 knockdown suppressed apoptosis and autophagy, accompanied by increased proliferation of glutamic acid-induced ICCs. More importantly, the effect of METTL3 knockdown on proliferation and autophagy was significantly reversed in glutamic acid-induced ICCs treated with LY294002 or GSK690693. Mechanistically, METTL3 deletion exerts its STC-repressive influence through the activation of the PI3K/AKT pathway. Collectively, the findings indicate that METTL3 modulates PI3K/AKT-mediated autophagy following LOP and highlight the potential of METTL3 as a therapeutic target in STC treatment.</p>