Postoperative ileus (POI) is the cessation or reduction of gastrointestinal (GI) motility after surgery. Reactive enteric glial cells (EGCs) are critical for maintaining bowel function. However, the triggering mechanisms and downstream effects of reactive EGCs in POI were poorly understood. The goal of this current study was to investigate whether the inducible nitric oxide synthase (iNOS)-driven reactive EGCs participated in GI motility disorders and mechanisms underlying altered GI motility in POI. Intestinal manipulation (IM)-induced POI mice and iNOS-/- mice were used in the study. Longitudinal muscle and myenteric plexuses (LMMPs) from the distal small intestine were stained by immunofluorescence. Our results found that the GI motility disorders occurred in the IM-induced POI mice, and reactive EGCs were observed in LMMPs. Glial metabolic inhibitor gliotoxin fluorocitrate (FC) treatment or iNOS gene knockout attenuated GI motility dysfunction. In addition, we also found that FC treatment or iNOS gene knockout significantly inhibited the fluorescence intensity macrophage colony-stimulating factor (M-CSF), which reduced M2 phenotype macrophages activation in LMMPs of IM-induced POI mice. Our findings demonstrated that iNOS-driven reactive EGCs played a key role and were tightly linked to the MMs homeostasis in the POI mice. EGCs are emerging as a new frontier in neurogastroenterology and a potential therapeutic target.Copyright © 2023. Published by Elsevier B.V.